Fluorescence Imaging Apllications Guide

Fluorescence
Imaging
Applications
Guide
Molecular Dynamics FluorImager (FI) and Storm Compatibility
with Selected Fluorescence Applications
Application/Label Vendor FI 575/SI FI 595 Storm 840 Storm 860

Nucleic Acids in Gels
Ethidium Bromide (Many) X X
SYBR Gold Molecular Probes X X X X
SYBR Green I Molecular Probes X X X X
Vistra Green Amersham Pharmacia Biotech X X X X
SYBR Green II Molecular Probes X X X X
SYTO 61 Molecular Probes X
Fluorescence 5' Oligo Kit Amersham Pharmacia Biotech X X X X
Nucleic Acids in Microplates
PicoGreen Molecular Probes X X X X
RiboGreen Molecular Probes X X X X
Nucleic Acids on Membranes
AlkPhos Direct Amersham Pharmacia Biotech X X X X
ECF RP Labeling Amersham Pharmacia Biotech X X X X
ECF 3' Oligo Kit Amersham Pharmacia Biotech X X X X
Chemifluorescent Substrates
ECF Substrate/AttoPhos Amersham Pharmacia Biotech X X X X
DDAO-Phosphate Molecular Probes X
ECL Plus Amersham Pharmacia Biotech X X X X
Protein in Gels
SYPRO Orange Molecular Probes X X X X
SYPRO Red Molecular Probes X X
Protein on Membranes
ECF Western Kit Amersham Pharmacia Biotech X X X X
ECF Western Blotting Amersham Pharmacia Biotech X X X X
Reagent Pack
PBXL-3 Antibodies Amersham Pharmacia Biotech X X X X
Protein in Microplates
NanoOrange Molecular Probes X X X X
Multipurpose Labels
Fluorescein X X X X
Cy3 Amersham Pharmacia Biotech X X
Cy5 Amersham Pharmacia Biotech X
FluorImager/Storm Operations
T
he purpose of this guide is to provide the basic information necessary to 2
make the most of the fluorescence imaging capabilities of the Molecular Fluorescence Imaging Users Group 2
Dynamics fluorescence scanning systems: FluorImager™ and Storm™. The Nucleic acids in gels
icons at the top of each page indicate which of these systems are compatible with
Ethidium Bromide 3
the application. A bold icon indicates that the scanner is fully compatible with SYBR® Gold 5
the application; a gray icon indicates some compatibility with the application SYBR® Green I 7
described on the page. ™
Vistra Green 9
SYBR® Green II 11
The information will allow the user to achieve quantitative, high-resolution SYTO® 61 13
images of gels, blots and microplates without exposure to film or storage Fluorescence 5' Oligolabeling Kit 15
phosphor screens, and to take positive and effective steps toward reducing the use Nucleic acids in microplates
of radiation in the lab. PicoGreen™ 17
RiboGreen™ 19
In the first section, applications are presented in an easy-to-understand format
covering specific and non-specific detection of nucleic acids and proteins in gels, Nucleic acids on membranes
membranes and microplates. These protocols are field-proven and have worked AlkPhos Direct™ 21
reliably in customers’ laboratories as well as our own. The procedures are printed ECF™ Random Prime Labeling 23
and Signal Amplification Kit
in a convenient one-page format that can be referred to at the bench.
ECF™ 3' Oligolabeling and 25
The second section is a more comprehensive list of dyes, the spectral Signal Amplification Kit
characteristics of which are compatible with the FluorImager and Storm. Some Chemifluorescent substrates
of these dyes have not been rigorously tested in our lab. ECF™ Substrate or AttoPhos® 27
DDAO-Phosphate 29
In addition to the applications presented in this Guide, technical information on ECL Plus™ Western Substrate 31
specialized fluorescent applications such as western blotting, two-color imaging Protein in gels
and gel-shift assays is available from Molecular Dynamics. For more information
SYPRO™ Orange 33
on these other specific applications, please contact us by calling SYPRO™ Red 35
(800) 333-5703 or visit our web site at www.mdyn.com. An electronic bulletin
Protein on membranes
board is available for users to discuss fluorescent applications (see page 2 for details).
ECF™ Western Blotting Kit 37
ECF™ Western Blotting Reagent 39
Pack (see also ECL Plus Substrate)
PBXL-3 Antibodies 41
(see also Cy5, Fluorescein, Cy3)
Protein in microplates
NanoOrange® 43
Multipurpose labels
Fluorescein 45
Cy™5 47
Cy™3 49
Additional Fluorochromes 51
The Molecular Dynamics

FluorImager system scans
samples in just minutes.
Powerful ImageQuant™ software gives fast quantitative results.
The Molecular Dynamics Storm system for gel and

blot analysis unites proven PhosphorImager™ system
technology with two non-radioactive fluorescent
imaging modes.
1
FluorImager™/Storm™ Fluorescence Imaging
Operations Users Group
1. Filter Molecular Dynamics operates an electronic bulletin board to help users
of Molecular Dynamics fluorescence scanning systems find, share and
In Storm, the appropriate filters are selected automatically for each scan discuss application hints, tricks and problems. This bulletin board
mode. With the FluorImager, the user selects an emission filter based on functions as a “mail exploder”–messages sent to fluorusers@mdyn.com
the excitation source used and the application. Imaging with the will be forwarded automatically to all members. Current members
488nm excitation line of the FluorImager does not require emission include FluorImager and Storm users, Molecular Dynamics applications
filtering for single-color applications (an internal long-pass filter blocks scientists, and scientists from other companies.
excitation light and passes light at 515nm and above). The investigator
is left with a choice related to optimizing the signal-to-noise ratio.
Filtering may decrease the background levels, but will also decrease To join:
signal from the dye. This could, for example, be the case when using a
Send a message to fluorusers@mdyn.com. In the body of the message
570DF30 emission filter for detection of an ECF Southern blot.
include the word “Subscribe,” your name and email address.
Recommendations for emission filters are found in the Instrument
Setup section for each fluorescent label or dye in this guide. Choose
appropriate filters for multicolor applications according to the peak To send a message:
fluorescent emission for each label used.
Email your message to fluorusers@mdyn.com. Please make the subject
as descriptive as possible to help everyone deal with the large volume
Note that excitation with the 514nm source of the FluorImager 595 of incoming messages.
does require the user to select a suitable drop-in emission filter before
initiating a scan.
2. Photomultiplier Tube (PMT)

The PMT voltage recommended in each case is a good starting point
from which further optimization may be possible. Sensitivity versus
PMT voltage varies for different instruments. The optimum voltage
needed will differ for each experiment, based upon sample concentra-
tion, sample media and thickness. For quantitative results, always check
to see if any pixels are saturated before proceeding with data interpreta-
tion. Refer to the ImageQuant software manual. If pixel saturation
occurs, rescan with a lower PMT setting. Optimal signal-to-noise ratio
is usually achieved over a fairly wide range, increasing the PMT voltage
does not improve sensitivity.
3. Sensitivity
Normal sensitivity is used for most scans. Selecting the high sensitivity
setting may increase the signal-to-noise ratio by scanning eight times
per pixel and averaging. This setting does not affect the file size.
4. Pixel Size
200µm is the lowest resolution setting and sufficient for most scans. If
higher resolution is required, select a smaller pixel size in Scanner
Control. This will increase the scan time and file size.
5. Calibration
The purpose of calibration on the FluorImager is to correct for errors
caused by lack of uniformity in light collection along the x-dimension.
2
Ethidium Bromide (EtBr)
Benefits
© More sensitive on the FluorImager system than on a UV transillu-
minator
© Easy, inexpensive to use
© Detects 200pg/band dsDNA in agarose or acrylamide, including
denaturing gels
© Detects ~10ng/band RNA in non-denaturing agarose gels
© Greater dynamic range of the FluorImager system over Polaroid®
film allows visualization and quantitation of both weak and dark
bands in the same scan
© Rapid quantitative analysis using the FluorImager system
Uses
Gel Shift Assay–post-stained with ethidium bromide and imaged with
Staining of dsDNA and RNA in gels FluorImager
Image courtesy of Dr. Velleman, Max-Planck Institute for Molecular

Excitation Maximum Genetics, Berlin.
526nm
Emission Maximum
605nm
Post-Staining
© Run gel
© Stain with 0.5µg/ml EtBr in water, TE, or TBE for at least 20
minutes with shaking
© Destain for 20 minutes in water
Gel Casting with EtBr

© Cast gel with final concentration of 0.25 to 0.5µg/ml EtBr
© Destain for 20 minutes in water (optional)
3 Nucleic Acids in Gels

Tips Detection of single-stranded nucleic acids is less sensitive than
detection of double-stranded DNA. Exact figures for ssDNA and RNA
© TE, TAE, TBE, or water can be used to make up the EtBr staining
may not be available. Band sharpness and band density will affect limit
solution.
of detection. Compared to agarose, sensitivity in a polyacrylamide gel
© EtBr does not have a high enough binding constant to DNA to will be higher due to its relatively low background fluorescence. Better
make pre-staining a viable option. sensitivity in agarose gels is achieved by pouring thinner and lower
© Binding efficiency and sensitivity of EtBr with ssDNA and RNA is percentage gels. The following detection limits are for gels that have
less than with dsDNA. been stained after electrophoresis (post staining) and are approximate.
Detection limits in gels that have been cast with the stain and for
© EtBr and ™Vistra Green compete for DNA binding. Staining first samples that have been stained (pre-electrophoresis) may be 3- to 5-
with EtBr, then with Vistra Green will give better results than with fold lower. The PMT voltage settings are suggested and the optimal
EtBr alone, but inferior to pure Vistra Green staining. setting will depend on sample concentration.
© For use in agarose and polyacrylamide gels. To stain denaturing gels,
wash gel briefly to remove urea before staining. Instrument Setup
© Longer stain times do not improve sensitivity. However, when FluorImager
dealing with highly concentrated, tightly packed bands in a high Excitation 488nm
percentage gel, allow more time for the EtBr to penetrate further Emission Filter 610RG
into the bands to improve visualization. PMT Voltage 700 to 800
© Staining for less than 20 minutes is not advised.
© Destaining for longer than 20 minutes can erase band signal Detection Limits DNA ssDNA/RNA
strength. FluorImager (pg/band) (pg/band)
© Casting gels with EtBr saves time after electrophoresis, but may also Agarose 200 10,000
alter migration of fragments. EtBr migration causes non-uniform Polyacrylamide 100
background signal in those gels. Post-staining should be used for
quantitative analysis.
Linear Range of Detection
© Store dye solution covered with aluminum foil to prevent bleaching
of fluorophore by overhead lighting. FluorImager ~50-fold
© Stain solution can be reused when stored at 4°C, but must be
strengthened by addition of fresh EtBr stock solution to compen-
sate for that taken out of solution by the staining of previous gels. Storage
© Diluted stain solution will keep up to one month at 4°C. Powder can be stored at room temperature but protected from light.
© It is not advisable to do either restriction digests or blotting after In aqueous form, the stain should be stored at 4°C and protected from
EtBr staining. light.
Binding Handling
EtBr is a non-covalent intercalating dye. Use gloves due to mutagenicity and dispose of properly.
Vendor
There are many sources of EtBr.
4
SYBR® Gold
Benefits
© At least 10X more sensitive on the FluorImager system than EtBr
on an UV transilluminator
© Allows post-stain processing of DNA, e.g., restriction digest or
Southern blotting
© Replaces silver staining and radioactive labeling in some applica-
tions, e.g., SSCP
© Effective with both glyoxal and formaldehyde RNA gels
© Detects about 40pg/band in agarose or acrylamide, even
denaturing gels
© Quick quantitative analysis using the FluorImager or Storm system
DNA markers in agarose gel stained with SYBR Gold and
imaged with FluorImager.
Uses
The primary use of SYBR Gold is for staining DNA and RNA in
agarose and acrylamide gels. SYBR Gold can be used in urea, glyoxal
and formaldehyde gels.
Excitation Maximum
495nm
Emission Maximum
537nm
Post-Staining
© Run gel.
© Use centrifuge to spin stock solution to bottom of tube. Dilute
SYBR Gold to a final dilution of 1:10,000 in TE, TBE, or TAE (pH
7.0 to 8.5).
© Leave gel to shake in dye solution for 10-30 minutes, longer for
thicker or higher percentage gels.
© No destaining necessary.
Pre-Staining
(preferred method for high percentage gels) DNA markers in agarose gel (same as above)
stained with SYBR Gold and imaged with Storm.
© Incubate DNA with 1:5000 dilution of SYBR Gold for 15 minutes
prior to electrophoresis. Add loading dye after the 15 minute
incubation.
Casting Gel with SYBR Gold

© Dilute SYBR Gold 1:10,000 into cooled gel solution just prior to
casting.

© Use double-gloved protection against DMSO stock solution.
© Cover dye solution with aluminum foil to prevent bleaching of of detection. Compared to agarose, sensitivity in a polyacrylamide gel
fluorophore by overhead lighting. will be higher due to its relatively low background fluorescence. Better
© SYBR Gold and EtBr compete for DNA binding. Staining first sensitivity in agarose gels is achieved by pouring thinner and lower
with EtBr, then with SYBR Gold will give better results than with percentage gels. The following detection limits are for gels that have
EtBr alone, but inferior to pure SYBR Gold staining. been stained after electrophoresis (post staining) and are approximate.
© Avoid use of bind silane with SYBR Gold on polyacrylamide gels: samples that have been stained (pre-electrophoresis) may be 3- to 5-
it is preferable to use a Gel-Slick-type of product on the opposite fold lower. The PMT voltage settings are suggested and the optimal
plate. setting will depend on sample concentration.
© Staining of extra-large sandwich gels can be easily accomplished by
laying the gel sandwich onto paper towels, having cleaned the glass Instrument Setup
plates well first. Remove top glass plate, then pipette enough
Fluor Imager
1:10,000 SYBR Gold stain onto gel to cover completely. Spread
Excitation 488 nm
evenly with a Pasteur pipette and leave protected from light for 10
Emission Filter 530DF30
to 60 minutes.
PMT Voltage 600 to 800
© If required, SYBR Gold can be removed from dsDNA by simple Storm
ethanol precipitation. Bring solution up to 100mM NaCl, add 2.5 (Blue Fluorescence)
volumes 95% EtOH, incubate 20 minutes on ice, centrifuge 10 PMT Voltage 700 to 900
minutes at 4°C.
© If covered and refrigerated, stain solution is good for three to four
Detection Limits DNA ssDNA/RNA
days.
FluorImager (pg/band) (pg/band)
Agarose 40
Binding Polyacrylamide 10 300
SYBR Gold is thought to bind non-covalently to the phosphodiester Storm
backbone of nucleic acids. Agarose 500
Polyacrylamide 40

FluorImager ~100-fold
Storm ~50 to 100-fold
Storage
In DMSO in plastic, protected from light.
Stock solution kept at –20°C is stable for six to twelve months.
Diluted reagent kept at 4°C is stable for three to four days.
Handling
Treat as a potential mutagen, as data is unavailable.
Vendor
Molecular Probes, Inc.
4849 Pitchford Avenue
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
Technical assistance: 541.465.8353
6
SYBR® Green I
Benefits
on a UV transilluminator.
Southern blotting.
tions, e.g., SSCP.
© No need to destain, because of high signal from DNA-bound dye
© Detects about 40pg/band in agarose or acrylamide, even denaturing
gels
© Quick quantitative analysis using the FluorImager or Storm system.
Uses
The primary use of SYBR Green I is for staining dsDNA. Single
stranded DNA and RNA can also be detected with SYBR Green I,
but with a 5 to 20-fold decrease in sensitivity. SYBR Green I is a
superior substitute for EtBr in most nucleic acid applications. SYBR
Green I does not inhibit the activity of most restriction enzymes or
interfere with Southern transfer. Triplex microsatellite analysis using SYBR Green I and imaged using FluorImagerSI.
Excitation Maximum
497nm
Emission Maximum
520nm
Post-Staining
© Run gel.
SYBR Green I to a final dilution of 1:10,000 in TE, TBE, or TAE
(pH 7.0 to 8.5).
© No detaining necessary.
Pre-Staining
(preferred method for high percentage gels)
DNA restriction digest pre-stained with
© Incubate DNA with 1:5000 dilution of SYBR Green I for 15
SYBR Green I, diluted five-fold serially,
minutes prior to electrophoresis. Add loading dye after the 15 resolved in an agarose gel and scanned
minute incubation. on Storm.
Casting Gel with SYBR Green I

© Dilute SYBR Green I to 1:10,000 into cooled gel solution just
prior to casting.

© SYBR Green I and EtBr compete for DNA binding. Staining first sensitivity in agarose gels is achieved by pouring thinner and lower
with EtBr, then with SYBR Green I will give better results than percentage gels. The following detection limits are for gels that have
with EtBr alone, but inferior to pure SYBR Green I staining. been stained after electrophoresis (post staining) and are approximate.
© Avoid use of bind silane with SYBR Green I on polyacrylamide samples that have been stained (pre-electrophoresis) may be 3- to 5-
gels: it is preferable to use a Gel-Slick-type of product on the fold lower. The PMT voltage settings are suggested and the optimal
opposite plate. setting will depend on sample concentration.
© Staining of extra-large sandwich gels can be easily accomplished by
laying the gel sandwich onto paper towels, having cleaned the glass Instrument Setup
1:10,000 SYBR Green I stain onto gel to cover completely. Spread Fluor Imager
evenly with a Pasteur pipette and leave protected from light for 10 Excitation 488 nm
to 60 minutes. Emission Filter 530DF30
© If required, SYBR Green I can be removed from dsDNA by simple
Storm
ethanol precipitation. Bring solution up to 100mM NaCl, add 2.5
(Blue Fluorescence)
volumes 95% EtOH, incubate 20 minutes on ice, centrifuge 10
minutes at 4°C.
© Pre-staining and precasting gels with SYBR Green I may produce
poorer sensitivity than post-staining in low percentage gels and can Detection Limits DNA ssDNA/RNA
also result in anomalous band migration. However, sensitivity can
FluorImager (pg/band) (pg/band)
be improved using these methods with gels containing >2%
Agarose 40
agarose.
Polyacrylamide 10 300
© Avoid use of SYBR Green I that has been through more than four Storm
freeze/thaw cycles. Agarose 500
Polyacrylamide 40

Binding
SYBR Green I is thought to bind non-covalently to the Storm ~50 to 100-fold
phosphodiester backbone of nucleic acids.
Storage
Diluted reagent kept at 4°C is stable for three to four days.
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
8
™
Vistra Green
Benefits
on an UV transilluminator.
Southern blotting.
tions, e.g., SSCP.
© Detects about 40pg/band in agarose or acrylamide, even denaturing
gels
© Quick quantitative analysis using the FluorImager or Storm system.
Uses
The primary use of Vistra Green is for staining dsDNA. Single stranded
DNA and RNA can also be detected with Vistra Green, but with a 5 to
20-fold decrease in sensitivity.Vistra Green is a superior substitute for
EtBr in most nucleic acid applications.Vistra Green does not inhibit the
activity of most restriction enzymes or interfere with Southern transfer.
Excitation Maximum
490nm (secondary peak at 254nm)
Emission Maximum
Agarose gel post-stained with Vistra Green and imaged with FluorImager.
520nm
Post-Staining
© Run gel.
Vistra Green to a final dilution of 1:10,000 in TE, TBE, or TAE
(pH 7.0 to 8.5).
© No detaining necessary.
Pre-Staining
(preferred method for high percentage gels)
© Incubate DNA with 1:5000 dilution of Vistra Green for 15

minutes prior to electrophoresis. Add loading dye after the 15
minute incubation.
Casting Gel with Vistra Green

© Dilute Vistra Green 1:10,000 into cooled gel solution just prior to
casting.

© Use double-gloved protection against DMSO stock solution
© ™Vistra Green and EtBr compete for DNA binding. Staining first sensitivity in agarose gels is achieved by pouring thinner and lower
with EtBr, then with Vistra Green will give better results than with percentage gels. The following detection limits are for gels that have
EtBr alone, but inferior to pure Vistra Green staining. been stained after electrophoresis (post-staining) and are approximate.
© Pre-staining and pre-casting gels with Vistra Green produce poorer samples that have been stained (pre-electrophoresis) may be 3- to 5-
sensitivity than post-staining in low percentage gels, and can also fold lower. The PMT voltage settings are suggested and the optimal
result in anomalous band migration. However, for gels containing setting will depend on sample concentration.
>2% agarose, sensitivity can be improved using these methods.
© If required sensitivity is not obtained, leave gel in Vistra Green Instrument Setup
overnight, but only if absolutely necessary, as this can cause band Fluor Imager
diffusion. Excitation 488nm
© Avoid use of bind saline with Vistra Green on polyacrylamide gels; Emission Filter 530DF30
it is preferable to use a Gel-Slick-type of product on the opposite PMT Voltage 600 to 800
plate. Storm
© Staining of extra-large sandwich gels can be easily accomplished by (Blue Fluorescence)
laying the gel sandwich onto paper towels, having cleaned the glass PMT Voltage 700 to 900
1:10,000 Vistra Green stain onto gel to cover completely. Spread Detection Limits DNA ssDNA/RNA
evenly with a Pasteur pipette and leave protected from light for 10
to 60 minutes. FluorImager (pg/band) (pg/band)
Agarose 40
© If required,Vistra Green can be removed from dsDNA by simple
Polyacrylamide 10 300
ethanol precipitation. Bring solution up to 100mM NaCl, add 2.5
volumes 95% EtOH, incubate 20 minutes on ice, centrifuge 10 Storm (pg/band)
minutes at 4°C. Agarose 500
Polyacrylamide 40
© If covered and refrigerated, stain solution is good for three to four
days.
© Stain solution can be reused three or four times when properly Linear Range of Detection
stored.
© Vistra Green has been tested for stability through ten freeze/thaw Storm ~50 to 100-fold
cycles. Make sure the contents of the vial thaw completely before
removing any material because the effective concentration of the
dye will be diminished if material is withdrawn before complete Storage
thawing. In DMSO in plastic, protected from light.
Binding Diluted reagent kept at 4°C is stable for three to four days.
Vistra Green is thought to bind non-covalently to the phosphodiester
backbone of nucleic acids.
Handling
Treat as a potential mutagen as data is unavailable.
Vendor
Amersham Pharmacia Amersham Pharmacia
Biotech, Inc., USA Biotech AB, Sweden
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Amersham Pharmacia www.apbiotech.com
Biotech Ltd., England
Tel: +44 1494 544000
10
SYBR® Green II
Benefits
© Up to 5X more sensitive than EtBr on a UV transilluminator
© Allows post-stain processing of DNA, e.g., northern blotting
© Simple post-staining replaces labeling, e.g., differential display gels
© Can also be used under denaturing conditions, e.g., formaldehyde
gels
Uses
Staining of RNA in agarose and polyacrylamide gels. Superior
substitute for EtBr. Does not interfere with northern transfer.
Excitation Maximum
497nm (secondary peak at 254nm)
Emission Maximum RNA molecular weight markers stained

with SYBR Green II and imaged on
513nm FluorImager
Post-Staining
© Run gel.
© No wash step necessary for urea or formaldehyde in denaturing
gels.
© Dilute SYBR Green II to a final concentration of 1:10,000 in TE,
TBE or TEA for non-denaturing and denaturing polyacrylamide
gels; 1:5,000 for agarose/formaldehyde gels.
© Leave gel to shake in dye solution for 10 to 60 minutes, longer for
© No destaining step necessary.

© SYBR Green II and EtBr compete for DNA binding. Staining first sensitivity in agarose gels is achieved by pouring thinner and lower
with EtBr, then with SYBR Green II will give better results than percentage gels. The following detection limits are for gels that have
with EtBr alone, but inferior to pure SYBR Green II staining. been stained after electrophoresis (post-staining) and are approximate.
© Washing out of formaldehyde from agarose gels prior to staining samples that have been stained (pre-electrophoresis) may be 3- to 5-
gives better results, but is not required. fold lower. The PMT voltage settings are suggested and the optimal
© Avoid repeated freeze/thawing of SYBR Green II stock solution setting will depend on sample concentration.
© If refrigerated, diluted staining solution is stable for one week.
Instrument Setup
© Stain can be reused three or four times when properly stored.
© SYBR Green II will also stain DNA, but usually gives a weaker FluorImager
signal than Vistra Green, and it results in a higher background. Excitation 488nm
Binding Storm
(Blue Fluorescence)
SYBR Green II is thought to bind non-covalently to the
phosphodiester backbone of the DNA or RNA molecule.
Detection Limits RNA

FluorImager (ng/band)
Agarose 10
Storm
Agarose 100

Storage
Stock solution kept at –20°C keeps for six to twelve months.
Diluted reagent kept at 4°C keeps one week in TE/TAE, but only 24
hours in water.
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
12
SYTO® 61
Benefits
© Nucleic acid stain for red fluorescence mode of Storm 860.
© As sensitive as SYBR Gold, SYBR Green, and Vistra Green
detection in the blue fluorescence mode of Storm.
tions.
© Quick quantitative analysis using the Storm 860 system.
Uses
The primary use of SYTO 61 Green 1 is for staining nucleic acids in
gels.
Excitation Maximum
630nm
Emission Maximum DNA ladder diluted two-fold serially, run in an agarose gel, stained
with SYTO 61 and imaged with Storm 860.
645nm
Post-Staining
© Run gel.
SYTO 61 500-fold in ethanol and then 10-fold in TE buffer giving
a final dilution of 1:5000 in 10% ethanol.
© Leave gel to shake in dye solution for 20 to 45 minutes, longer for
© Rinse in 10% ethanol to decrease background.

Tips Instrument Setup
© Use double-gloved protection against DMSO stock solution. Storm
© Cover dye solution with aluminum foil to prevent bleaching of
(Red Fluorescence)
fluorophore by overhead lighting. PMT Voltage 700 to 900
© SYTO 61 and EtBr may compete for DNA binding.

© Avoid use of bind silane with SYTO 61 on polyacrylamide gels: it
Detection Limits DNA
is preferable to use a Gel-Slick-type of product on the opposite Storm pg/band
plate, if needed. Agarose 500
Polyacrylamide 40
Binding
The SYTO dyes are hydrophobic, cell permeant nucleic acid stains and
are believed to bind to the phosphodiester backbone. Storm ~50 to 100-fold
Storage
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
14
Fluorescence 5’
Oligolabeling Kit
Benefits
© Any unmodified oligo with a free 5'-OH group can be labeled
using this method, eliminating the need for expensive fluorescein
phosphoramidites or lengthy protocols with amine coupling
© Probes stable at least six months at –20°C
© Typical yield after labeling and purification is at least 75%. This is
about 25% better yield than traditional coupling
© Reagent system specifically developed for quantitation on Molecu-
lar Dynamics fluorescence imaging systems
Detection of oncogenes with fluorescently labeled PCR primers on FluorImager.
© Allows direct in-gel detection and quantitation of bands
© No film exposure required Image courtesy of Dr. Parke Flick, Amersham Pharmacia Biotech.
Uses
PCR primer labeling
PCR product labeling
Gel shift assays
5' HO OH 3' + ATPγS
Excitation Maximum
T4 Polynucleotide Kinase Kinase Reaction
Fluorescein label 495nm 1 hour
S 37°C
_ =
–O – P– O OH 3'
Emission Maximum O–
5-IAF
Fluorescein label 520nm O O OH
COOH
5' Fluorescein
Method Labeled Oligo
NH—C—CH2l Coupling Reaction
O O OH 30 minutes
The following steps (described in detail in the protocol booklet =O 31°C
provided with kit) summarize oligolabeling:
COOH + Free 5-IAF
60 min Kinase reaction
30 min Coupling reaction NH—C—CH2
_
=
15 min Purification O S
_
(4 hr) Optional SurePure purification (only if unlabeled oligos must O = P_ O OH 3'

_
–O
be removed)
15 min Checking incorporation of fluorescein into DNA (rapid Purification
15 minutes
labeling assay) (30 minutes for optional
5' Fluorescein Labeled Oligo labeling efficiency step)
Total Time 2 hrs. 15 min.
The kit’s quick and easy labeling procedure takes just over two hours to
complete.

© Deviations from the protocol are strongly discouraged. FluorImager
© This labeling method uses a different fluorescein tag than the ECF
Excitation 488nm
Random Prime Labeling and ECF 3' Oligolabeling kits. The Emission Filter 530DF30
Fluorescence 5' Oligolabeling Kit label does not work with the PMT Voltage 600 to 800
ECF Signal Amplification Module. Storm
(Blue Fluorescence)
© A rotor with swinging buckets is required for spinning the PMT Voltage 900 to 1000
purification columns.
© If possible, unlabeled oligos should be dissolved in distilled water,
not in TE. Detection Limits
© When scanning a SurePure™ TLC plate on the FluorImager FluorImager (fmol/band)

(optional purification of the labeling reaction), the 610 long-pass Polyacrylamide 1 to 5
filter can be used to reduce plate background. Storm
Polyacrylamide 30 to 50
© Complete deblocking of the oligonucleotide is important to ensure
a free 5’-OH is available.
Storm ~100-fold
Binding
A single fluorescein molecule is attached to the 5' end of an oligo-
Storage
nucleotide.
5' oligolabeling reagents at –15 to –30°C.
Purifications columns at 2 to 8°C.
Kit contents stable at least three months under recommended
conditions.
Handling
Certain components of the kit are harmful. Use of gloves is advised
throughout.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
16
PicoGreen™
Benefits
© No need to run a gel for DNA quantitation.
© High throughput of at least 88 unknowns/plate, scanning three
plates simultaneously.
© Enables accurate quantitation of dsDNA in solution.
Uses
Quantitation of dsDNA in microplates.
Excitation Maximum
498nm
Emission Maximum
520nm
Method
© Dilute stock PicoGreen solution 1:200 in TE buffer.
© Add 50µl of PicoGreen into the required number of wells.
PicoGreen quantitation from the FluorImager.
© Add 50µl of calibration standards (adjacent table) to either row A or
column 1 of the microplate.
© Add 50µl of each unknown sample DNA to additional wells.
© Mix gently (avoid bubbles) using pipette.
© Incubate two to five minutes at room temperature.
Final Conc.
Sample DNA TE DNA (ng/ml)
1 2µl of a 198µl 10,000

1mg/ml stock sol.
2 70µl of #1 130µl 3,500
3 70µl of #2 130µl 1,125
4 70µl of #3 130µl 429
5 70µl of #4 130µl 150
6 70µl of #5 130µl 52.5
7 70µl of #6 130µl 18.4
8 70µl of #7 130µl 6.4
9 70µl of #8 130µl 2.25
10 70µl of #9 130µl 0.79
11 70µl of #10 130µl 0.27
12 0µl 130µl 0
17 Nucleic Acids in Microplates

© For calibration standards, use only DNA of precisely determined FluorImager
concentration. Do not use degraded DNA. Excitation 488nm
© If RNA contamination is above 1µg/ml, treat first with RNase
© Use clear, flat-bottomed, low-fluorescence microplates (Corning Storm
Costar). (Blue Fluorescence)
© When scanning on Storm, image quality is improved by using PMT Voltage 800 to 1000
microplate strips so that the wells sit flat on the scanning surface.
© If using Molecular Dynamics DNA Quantitation software, eight Detection Limits dsDNA
standards are required and must be placed in the first column of the
microplate. (ng/ml)
FluorImager 5
© To quantitate most accurately, use the Molecular Dynamics
Storm 50
ImageQuant software to draw an ellipse set within the inner wall of
Detection limits and dynamic range described above are achievable
one well and copy it to the other wells. Report Median value with
with up to 5mg/ml RNA contamination.
Background Correction set to None for quantitation.
© In Microsoft Excel, subtract the Median of the negative control
from each well. This is critical for good low-end linearity. Linear Range of Detection
© Prepare standard curve using DNA similar to the unknown, e.g., (ng/ml)
same size and source. FluorImager 5 to 3500
© PicoGreen has been used successfully after five freeze/thaw cycles.
Storm 100 to 3500
Binding Storage
No data available (non-covalent). Reagent is stable for six to twelve months kept in a protected container
at –20°C.
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
18
RiboGreen™
Benefits
© No need to run a gel for RNA quantitation.
© Enables accurate quantitation in solution in microplate.
© Linearity is maintained in the presence of several contaminating
components (i.e., nucleotides, proteins, salt)
Uses
Quantitation of RNA in solution
Excitation Maximum
500nm
Emission Maximum
520nm
Method
© Two different dye concentrations are required to achieve the full
linear dynamic range of this assay. Both a high range assay (20ng/ml
to 1µg/ml RNA) and a low range assay (1ng/ml to 50ng/ml
RNA) are possible. These require different dilutions of the RiboGreen quantitation using the FluorImager.
RiboGreen reagent (1:200 and 1:2000, respectively).
© Nuclease-free TE (10mM Tris-Cl, 1mM EDTA, pH 7.5) is used as
the assay buffer. Prepare the RiboGreen reagent by diluting either
200-fold or 2000-fold with TE (7.5), depending on the assay.
© Dilute RNA (if needed) in nuclease-free TE (7.5). See adjacent
table for preparing high and low range standard curves: use
Preparation of RNA Standards
precisely assayed RNA as a standard. Add 100µl of each RNA
sample to wells of a microplate. High Range Standards
© Add 100µl of diluted RiboGreen reagent to each well. Volume (µl) Volume (µl) Final RNA
© Mix gently (avoid bubbles) with pipette. TE 2µg/ml RNA Conc. (ng/ml)
© Incubate two to five minutes. 0 100 2000
50 50 1000
90 10 200
98 2 40
100 0 Blank
Low Range Standards

Volume (µl) Volume (µl) Final RNA
TE 100ng/ml RNA Conc. (ng/ml)
0 100 100
50 50 50
90 10 10
98 2 2
100 0 Blank
19 Nucleic Acids in Microplates

© Use double-gloved protection against DMSO stock solution. FluorImager
© Cover dye solution with aluminum foil to prevent bleaching of
Excitation 488nm
fluorophore by overhead lighting. Emission Filter 530DF30
© All solutions should be prepared in sterile disposable plasticware or Storm
nuclease-free glassware. (Blue Fluorescence)
© When possible, prepare more than one dilution of unknowns. PMT Voltage 800 to 1000
© RiboGreen also binds DNA. Pretreatment of mixed samples with
DNAse can be used to generate an RNA-selective assay. Detection Limits RNA
© To quantitate most accurately, use the Molecular Dynamics
(ng/ml)
ImageQuant software to draw an ellipse set within the inner wall of
FluorImager 1
the well and copy it to the other wells. Set Background Correction
Storm 10
set to None and report Median pixel values. Save your template to
avoid redrawing ellipses for future scans.
© In Microsoft Excel, subtract the Median pixel intensity of the blank
from the Median value of the sample. (ng/ml)
© Prepare standard curve using RNA similar to the unknowns, e.g.,
FluorImager 1 to 1000
source, contaminants. Storm 10 to 1000
Storage
Ribosomal RNA standard is kept at 4°C.
RiboGreen reagent is kept at –20°C protected from light
20X TE is kept at room temperature.
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
20
AlkPhos Direct™
Benefits
© Direct covalent labeling of DNA or RNA probe with thermostable
alkaline phosphatase.
© No hapten used for labeling, so immunodetection is not needed.
© Following hybridization and stringency washes, detection is direct.
© Labeling and detection protocol designed specifically for use with
Molecular Dynamics fluorescence scanning systems.
© Probes stable for up to six months at –20°C.
Uses
Southern blots (single copy gene detection)
Northern blots (of moderate to high copy mRNA)
Dot and slot blots
DNA fingerprinting
Colony and plaque lifts
Excitation Maximum
ECF substrate 440nm
Emission Maximum
ECF substrate 560nm
Human genomic Southern (bcl-2 target)
detected with ECF Substrate and Storm
Method scanning.
Day 1 Day 2 Day 3 Day 4

Preparing Blot Labeling Signal Amplification Scanning
2 to 3 hr Electrophoresis 30 min Fixing DNA to 30 min Stringency washes 5 min Scanning

membrane
Overnight Blotting 30 to Probe labeling
120 min
15 min Prehybridization
2 to 16 hr Hybridization 1 to 16 hr Development with
ECF substrate
21 Nucleic Acids on Membranes

© Use purified insert as probe when possible. FluorImager
© If not used immediately, keep labeled probe on ice (up to two hours) Excitation 488nm
or for long-term storage, add glycerol to 50% and place at -20°C. Emission Filter 570DF30
© Increasing the labeling reaction incubation time from 30 minutes to Storm
2 hours at 37°C may improve sensitivity. (Blue Fluorescence/Chemifluorescence)
© Use 10-20ng/ml labeled probe in hybridization. PMT Voltage 600 to 700
© Single-stranded nucleic acids smaller than 50 nucleotides may be
labeled, however probes greater than 300 nucleotides are recom- Detection Limits
mended for single copy gene detection.
FluorImager 0.25 pg target in 0.5 µg human genomic
© Do not denature the probe before use.
DNA
© Note: there is no protocol for assessing probe labeling efficiency. Storm 0.25 pg target in 0.5 µg human genomic
DNA
Binding
Thermostable alkaline phosphatase is cross-linked covalently to the
target nucleic acid using formaldehyde. Probe labeling with this kit is FluorImager ~50-fold
believed to result in average attachment of one alkphos group per 10 to Storm ~50-fold
20 nucleotides.
Storage
Hybridization buffer and blocking reagent at 15 to 25°C, all other
components at 2 to 8°C. Stable for three months when stored under
recommended conditions.
Handling
Certain components of this kit are harmful, including sodium azide and
formaldehyde. Wear gloves and suitable protection at all times.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
22
ECF™ Random Prime Labeling
and Signal Amplification Kit
Detection of p53 oncogene
Benefits in a Southern blot of mouse
genomic DNA
© No probe purification step necessary.
© Fluorescein as a hapten allows direct visual confirmation of probe
labeling efficiency, which gives confidence to proceed.
© Probes stable at least six months at –20°C.
lar Dynamics fluorescence scanning systems.
© Eliminates the need for darkroom, film and chemiluminescent
screens.
© Greater dynamic range of Molecular Dynamics fluorescence
scanning systems over film, allows visualization and quantitation of
light and dark bands in the same scan.
© Direct detection of fluorescein option for high target applications.
Excitation Fluorescence Indirect Detection
550-570nm
Uses The ECF Signal
Substrate
Amplification Module
Southern blots (single copy gene detection using probe >400bp) Fluorophore
boosts sensitivity by
coupling alkaline
Northern blots (of moderate to high copy mRNAs) Alkaline phosphatase to the
Phosphatase
Dot and slot blots Phosphate fluoresceinated DNA
Group
Anti-Fluorescein probe. Alkaline phospha-
DNA fingerprinting Probe
tase catalyzes the forma-
Colony and plaque lifts Fluorescein tion of stable fluorophores
Target DNA which remain near the
probe and emit light when
Excitation Maximum Solid Support read in the FluorImager or
Storm systems.
Fluorescein label 495nm
ECF substrate 440nm
Laser
Excitation
Direct Detection
488nm For high-target applica-
Fluorescence
Emission Maximum 510-530nm tions, the fluoresceinated
DNA probe may be
Fluorescein label 520nm detected directly, without
enzymatic amplification.
ECF substrate 560nm
Probe
Fluorescein
Method
Target DNA
The following steps (described in detail in the protocol booklet Membrane
included with the kit) summarize the procedure for Southern and
northern blotting and detection:
2 to 3 hr Electrophoresis 30 min Fixing DNA to 30 min Stringency washes (for 5 min Scanning
membrane high expected signal, scan
for fluorescein at this stage)
Overnight Blotting 90 min Making randomly 1 hr Blocking
labeled DNA probe
30 min Checking incorpor- 1 hr Incubation with antibody
ation of fluorescein into
DNA (rapid labeling
assay)
30 min Prehybridization 30 min Washing
Overnight Hybridization 15 min to Signal development and
overnight detection

© Prepare blots early in the week for scanning and analysis later in the FluorImager
week. Excitation 488nm
Emission Filter 530DF30 – fluorescein
© Probes may be labeled and checked for incorporation of fluorescein
570DF30 - ECF
during electrophoresis and blotting.
© Blotting must be performed using positively-charged nylon Storm
membrane. (Blue Fluorescence/Chemifluorescence)
© Fixing by baking gives better results than fixing by UV cross- PMT Voltage 600 to 700
linking unless you use carefully controlled UV conditions, e.g., with (Be aware that high PMT voltage can result in membrane background
Amersham cross linker RPN 2500/2501. saturation.)
© Deviations from the protocol are strongly advised against; short cuts
employed with radioactive protocols should be avoided.
Detection Limits dsDNA
© Use of powder-free gloves is important, as the powder fluoresces.
FluorImager 0.25pg target in 0.5µg human genomic DNA
© Template DNA to be labeled must be at least 400bp long for Storm 0.25pg target in 0.5µg human genomic DNA
efficient labeling by this method. Otherwise use the ECF 3'
Oligolabeling Kit. Direct detection of fluorescein labeled probe bound to target is about
10- to 50-fold more sensitive on FluorImager than Storm (see pages
© For the signal amplification step, use containers and tips cleaned by 45-46 for similar comparison of direct fluorescein detection). Direct
sterilizing or by rinsing with 0.01M HCl or boiling water to detection of fluorescein may be possible with the FluorImager system
reduce background resulting from ambient alkaline phosphatase. for high target applications, such as screening of PCR products. Most
© Do not allow membrane to dry out during processing, or the applications will require signal amplification.
membrane cannot be stripped and reused.
© Always do an initial scan after developing the blot. High target
bands can develop within minutes, although single copy detection FluorImager ~50-fold
and most northerns require overnight development. Maximum Storm ~50-fold
signal is usually achieved within 24 hours incubation.
Storage
Binding
Labeling module at –15 to –30°C
Fluorescein comes covalently bound to dUTP in the kit. The kit
Signal Amplification Module at 2 to 8°C.
chemistry randomly incorporates fluoresceinated dUTPs during
complementary strand synthesis. Higher sensitivity is usually obtained
compared to 3' labeled probes where a limited number of labeled Handling
nucleotides are attached to the 3' end of the probe.
Certain components of the kit are harmful. Use of powder-free gloves
is advised throughout to prevent contamination of the blot and
reagents.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
24
ECF™ 3’ Oligolabeling and
Signal Amplification Kit
Benefits
© No probe purification step necessary.
© Fluorescein as a hapten allows direct visual confirmation of probe
labeling efficiency, which gives confidence to proceed.
© Probes stable at least six months at –20°C.
lar Dynamics fluorescence scanning systems.
screens.
a b
scanning systems over film allows visualization and quantitation of
weak and dark bands in the same scan. Southern blots of λ Hind III detected by a) random prime labeled λ Hind and b) by
a specific oligo (20mer) labeled with the Vistra ECF 3’ Kit. Total DNA loadings are
© Direct detection optional for high target applications. 10ng, 1ng, and 0.1ng per lane. Development times with the detection reagent
were a) two hours, b) overnight. Note the greater specificity possible with oligo
probes and the greater sensitivity of the random prime labeled probes.
Uses
This system is designed for labeling and detecting oligonucleotide
probes for:
Southern blots of high copy genes or low complexity genomes, such as
Excitation
bacterial DNA with probes <400bp 450 or 488nm
Fluorescence
540-560nm
Northern blots (having abundant message)

ECF Substrate
Dot and slot blots Fluorescent
Product
Labeling of oligos
Alkaline
Phosphate
Screening of PCR products Phosphatase
Group
Anit-fluorescein
Excitation Maximum
Probe

Target
Solid Support
ECF substrate 440nm
Emission Maximum The 3’ Oligolabeling Module attaches fluorescein to the 3’ end of the probe using
terminal deoxy-nucleotidyl transferase. There is no requirement to purify the prod-
Fluorescein label 520nm uct. Hybridization of probe to target DNA is performed as usual. The hybridization
stringency of the probe is not significantly affected by the fluorescein tail. Next,
ECF substrate 560nm block and incubate the blot with the anti-fluorescein-AP conjugate. Finally, develop
the blot with the ECF substrate and scan.
Method
The following steps summarize the procedure for Southern and high-
copy northern blotting and detection:
2 to 3 hr Electrophoresis 30 min Fixing DNA to 30 min Stringency washes (for 5 min Scanning
membrane high expected signal, scan
for fluorescein at this stage)
Overnight Blotting 90 min Labeling DNA probe 1 hr Blocking
30 min Checking incorpor- 1 hr Incubation with antibody
ation of fluorescein into
DNA (rapid labeling
assay)
30 min Prehybridization 30 min Washing
2 hrs to Hybridization 15 min to Signal development and
Overnight overnight detection

© This labeling method yields a detection limit at least ten-fold less FluorImager
sensitive than random prime labeling. It should be sensitive enough Excitation 488nm
for single copy detection of bacterial genomic DNA, but not Emission Filter 530DF30 – fluorescein
human. 570DF30 - ECF
© Probes can be labeled and checked for incorporation of fluorescein
during electrophoresis and blotting. Storm
(Blue Fluorescence/Chemifluorescence)
© Blotting must be performed using positively-charged nylon PMT Voltage 600 to 700
membrane.
(Be aware that high PMT voltage can result in membrane background
© Fixing by baking gives better results than fixing by UV cross- saturation.)
linking unless carefully controlled UV conditions are used, (e.g., the
Amersham cross linker RPN 2500/2501).
Detection Limits (ECF only)
© Deviations from the protocol are strongly discouraged. Short cuts
employed with radioactive blots should be avoided. FluorImager 10 attomoles of control oligo
© Use of powder-free gloves is important, as the powder fluoresces. Storm 10 attomoles of control oligo
© Always do an initial scan after developing blot. High target blots Ten attomoles control oligonucleotide using signal amplification. Fluo-
may develop within minutes, although single copy detection and rescein detection is about 10- to 50-fold more sensitive on FluorImager
most northerns may require overnight development. Maximum than Storm (see pages 45-46 for similar comparison of direct fluores-
signal is usually achieved by 24 hours incubation. cein detection). One femtomole of the labeled oligonucleotide probe
can be detected in a polyacrylamide gel using the FluorImager system.
© For signal amplification step, use containers and tips cleaned by
sterilizing or by rinsing with 0.01M HCl or boiling water to
reduce background resulting from ambient alkaline phosphatase. Linear Range of Detection
Storm ~50-fold
Binding
Multiple fluorescein molecules are covalently attached to the 3' end of
an oligonucleotide. The method will add up to 20 fluorescein-dUTPs Storage
in a 3' tailing reaction with terminal deoxynucleotidyl transferase. 3' Oligolabeling Module at –15 to –30°C
Signal Amplification Module at 2 to 8°C
Kit contents stable at least three months under recommended
conditions
Handling
Certain components of the kit are harmful. Use of powder-free gloves
reagents.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
26
ECF™ Substrate or AttoPhos®
Benefits
© Sensitivity comparable to chemiluminescence
screens
scanning systems over film allows visualization and quantitation of
weak and dark bands in the same scan
Uses
Substrate for alkaline phosphatase. Converts by enzymatic reaction to
fluorescent product, which emits light when excited by a suitable
excitation light source. Alkaline phosphatase-linked secondary
antibodies and ECF Substrate can be used to amplify signal detection
in a variety of microplate and membrane-based applications, such as
Southern, northern, western, dot and slot blots.
Excitation Maximum
440nm
Emission Maximum
560nm
Buffer
2.4M diethanolamine (DEA) in water Single copy RFLP blot probed with alkaline phosphatase labeled D4S139
0.23mM MgCl2 probe and developed for four hours with ECF Substrate.
pH to 10.0 using NaOH
Buffer is stable for one year when stored at 4°C.
Method
Use 1mM ECF Substrate dissolved in buffer, or use kit concentration
© For all procedures, including Southerns, westerns, and northerns,
use clean containers, pipette tips, etc., and either:
1. Pipette ECF Substrate onto plastic wrap and lay blot face down
onto solution. Be sure to coat blot completely and evenly with
substrate. Incubate as needed, then transfer blot face down to
scanning surface.
2. Place blot in plastic bag*, and add ECF Substrate solution. To
ensure even distribution, smooth with a pipette and squeeze out
excess liquid. Then, either scan face down or transfer to a clean
bag beforehand (particularly recommended for Southerns and
northerns).**
© Always scan immediately in case of rapid signal development, then
every 15 minutes up to one hour, then every hour, leaving
overnight if necessary.
* Suitable low fluorescence plastic bags are provided with the ECF Random Prime and 3' Kits.
Alternatively, use transparency sheets or Kapak® specimen bags for blot development. Stiffer bags,
such as Kapak bags (polyester bag made by 3M Corp.), may result in higher background, but
have the advantage of eliminating wrinkles.
** For western blots on PVDF membranes, blots can be scanned either wet or dry.
27 Chemifluorescent Substrates
© Always scan immediately. Some blots can develop within minutes, FluorImager
particularly western blots. Excitation 488 nm
© Use of water between scanning tray glass and plastic bag reduces Emission Filter 570DF30
optical refraction and results in a clearer image. PMT Voltage 500 to 600
Storm
© Use Mylar or a glass plate rather than plastic wrap as a cover. It is (Blue Fluorescence/Chemifluorescence)
less fluorescent. PMT Voltage 600 to 700
© Covered, refrigerated solution can be used for up to one month. (Be aware that high PMT voltage can result in membrane background
© Use of containers and tips cleaned by sterilizing or by rinsing with saturation.)
0.01M HCl or boiling water reduces background resulting from
ambient alkaline phosphatase.
Detection Limits
© Autoclaving blocking solution and filtering reagents can decrease
background from ambient alkaline phosphatase. FluorImager (Variable depending on application)
Storm (Variable depending on application)

Binding
FluorImager (Variable depending on application)
ECF Substrate is dephosphorylated to the fluorescent product, which Storm (Variable depending on application)
remains in the region in which it is produced. It is believed to adhere
to the membrane by charge interactions.
Storage
In clean glass or plastic, refrigerated and protected from light.
In buffer (2.4M DEA, 0.23m MgCl2 (pH 10.0))
Diluted reagent at 4°C keeps up to one week
Handling
Use powder-free gloves to protect against contamination from naturally
occurring alkaline phosphatase on hands.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
28
DDAO-Phosphate
Benefits
© Alternative substrate for alkaline phosphatase for use with Storm
860.
© Sensitivity comparable to chemiluminescence.
screens.
© Greater dynamic range of Molecular Dynamics Storm system over
film allows visualization and quantitation of weak and dark bands in
the same scan.
Uses
Red-excited substrate for alkaline phosphatase. Converts by enzymatic
reaction to the fluorescent product DDAO, which emits light when
excited by a suitable excitation light source. Use with any alkaline
phosphatase-linked secondary antibody in microplate and membrane-
based applications such as Southern, northern, western, dot and slot
blots.
Southern blot of EcoRI digested human placental DNA probed with bel-2
(2.8kb purified cDNA).
Excitation Maximum
646nm
Emission Maximum
660nm
Buffer
10mM Tris (pH 9.5)
1mM MgCl2
Method
© Dissolve DDAO-phosphate to 1.25 mg/ml in water
© Dilute 1:1000 in Tris/MgCl2 buffer (above)
© For all procedures, including Southerns, westerns, and northerns,

use clean containers, pipette tips, etc., and either:
1.Pipette DDAO-phosphate onto plastic wrap and lay blot face
down onto solution. Be sure to coat blot completely and
evenly with substrate. Incubate as needed, then transfer blot
face down to clean scanning surface.
2. Place blot in plastic bag*, and add DDAO-phosphate
substrate solution. To ensure even distribution, smooth with a
pipette and squeeze out excess liquid. Then, either scan face
down or transfer to a clean bag beforehand (particularly
recommended for Southerns and northerns).**
© Always scan immediately in case of rapid signal development, then
every 15 minutes up to one hour, then every hour, leaving
overnight if necessary.
* Suitable low fluorescence plastic bags are provided with the ECF Random Prime and 3’ Kits.
Alternatively, use transparency sheets or Kapak® specimen bags for blot development. Stiffer bags,
such as Kapak bags (polyester bag made by 3M Corp.), may result in higher background, but
have the advantage of eliminating wrinkles
** For western blots on PVDF membranes, blots can be scanned either wet or dry.
© Do not use diethanolamine buffer: it causes hydrolysis of DDAO- Storm
phosphate. (Red Fluorescence)
© Spin DDAO-phosphate stock solution for five minutes to eliminate
undissolved particles before diluting.
© Do a test scan immediately after adding substrate. Some blots can Detection Limits
develop within minutes. Storm (Variable depending on application)
© If scanning through a plastic bag, add a thin layer of water between Comparable to ECF
the glass scanning tray and the plastic bag to minimize optical
refraction artifacts.
© To prevent blots from drying out during scanning, cover with a
clean glass plate or Mylar (not plastic wrap). Storm (Variable depending on application)
© Background due to ambient alkaline phosphatase can be minimized
Comparable to ECF
by sterilizing containers and tips by autoclaving or rinsing in 0.01M
HCl, and by autoclaving all reagents. Storage
© Glove powder fluoresces; use powder-free gloves to handle blots.
Store stock solution at –20°C.
Binding
Handling
DDAO-phosphatase is dephosphorylated to DDAO by alkaline
No toxicity data available. Handle with gloves to prevent contamina-
phosphatase. DDAO remains localized on the membrane in the region
tion from ambient alkaline phosphatase.
of the target.
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
30
ECL Plus™ Western Substrate
Benefits
© Fluorescent detection of HRP (horseradish peroxidase)-based
westerns
© Improved quantitation compared to film detection
screens.
© Signal is stable for up to 12 hours
Uses
Western blotting
Excitation Maximum
~420nm
Emission Maximum
ß-tubulin detected with ECL Plus and imaged using
~460nm Storm (top) and FluorImager (bottom)
Method
© Prepare substrate (refer to manufacturer’s instructions)
© Following the last wash (after addition of the secondary antibody),
place blot protein side up on plastic wrap on a level surface and
apply reagent (100µl/cm2) so that the blot is completely covered.
© Incubate for five minutes.
© Carefully lift blot with clean forceps, briefly drain off excess reagent
and position in an open plastic bag or sheet protector.*
© Place the blot in the plastic sandwich sample side down onto the
glass. A small puddle of water may be used to form a tight interface
between the plastic and the glass. Work bubbles out by wiping back
of blot and cover with a glass plate to keep flat.
* Use sheet protector, transparency sheets or Kapak® specimen bags for blot development. Stiffer
bags, such as Kapak bags (polyester bag made by 3M Corp.), may result in higher background,
but have the advantage of eliminating wrinkles.
Tips
© Glove powder fluoresces; use powder-free gloves when handling Instrument Setup
blots and preparing to scan.
Fluor Imager
© Signal is stable for many hours, however, for best results, blot should
Excitation 488nm
be scanned within the first few hours after adding substrate. Emission Filter 530DF30
Imaging after overnight development can result in diffusion of PMT Voltage 500 to 700
signal. Storm
© Handle membrane carefully, using gloves and forceps at all times to (Blue Fluorescence/Chemifluorescence)
prevent membrane damage, which can result in areas of high PMT Voltage 600 to 800
background and spotting.
© Prepare working substrate fresh immediately before use.
Detection Limits
FluorImager (Storm detection is comparable to film
Binding and is 5-fold more sensitive than
Primary antibody binds specifically to protein of interest. Horseradish FluorImager)
peroxidase limited to secondary antibody converts ECL Plus substrate Storm
to a fluorescent product, which remains in region in which it is
produced. Linear Range of Detection
Storm ~20-fold
Storage
All components should be stored at 4°C. Components are stable for
three months when stored properly.
Handling
ECL Plus solution B contains ethanol and dioxane. Handle accordingly.
Wear gloves and safety glasses.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
32
SYPRO™ Orange
Benefits
© Simple, quick staining protocol.
© As sensitive as silver staining (~2ng/band for most proteins)
© Linear range from 5ng to at least 1µg.
© Allows western transfer and immunodetection after staining.
Uses
Nonspecific fluorescent staining of denatured proteins in 1D and 2D
SDS-polyacrylamide gels.
Excitation Maximum
472nm
Protein molecular weight markers stained with SYPRO Orange.

Emission Maximum
570nm
Method
© Run gel.
© Dilute SYPRO Orange 1:5000 in 7.5% acetic acid (enough to

cover gel). Mix well.
© Place gel and stain in a foil-covered container and shake gently for
30 to 60 minutes.
© For samples with high background, destain in 7.5% acetic acid for
five minutes.
© Rinse briefly in distilled water
Alternative Protocol for Western Transfer

Dilute SYPRO Orange 1:5000 in Tris/Glycine/Methanol transfer
buffer. Proceed with staining as above. If necessary, destain in transfer
buffer.
33 Protein in Gels
Tips Band sharpness and band density will affect limit of detection. The
following detection limits are for gels that have been stained after
© Ensure glass plates and staining containers are completely free of
electrophoresis (post staining) and are approximate. The PMT voltage
grease and fingerprints.
settings are suggested and the actual setting will depend on sample
© Use powder-free gloves to handle gels. concentration.
© Mix fresh staining solution immediately before using.
© Shake the staining solution vigorously before adding it to the gel. Instrument Setup
© Staining in acetic acid significantly impairs immunodetection. For FluorImager

western blotting, stain in transfer buffer (sensitivity may be slightly Excitation 488nm
diminished). Emission Filter 570DF30
© Staining solution can be reused effectively at least three times.
Storm
© When staining native gels, soak gels in 0.1% SDS (in either 7.5% (Blue Fluorescence)
acetic acid or water) for 30 to 60 minutes prior to staining. PMT Voltage 800 to 900
Sensitivity will be diminished in native gels.
© Methanol can adversely affect staining.
Detection Limits
© When staining acidic proteins, increase the concentration of SDS in (ng/band)
the gel and buffers to 0.2%. FluorImager 3
© Phospholipids will not be stained as effectively as other proteins. Storm 6
As for all protein staining systems, dye binding will depend on the
Binding amino acid composition of the proteins. Proteins in native gels will
exhibit more staining variability then proteins saturated with SDS.
Nonspecific, SDS-dependent binding to proteins.
Storm ~500-fold
Storage
Stock solution is stable for six months to one year stored at –20°C and
protected from light. Staining reagent diluted in buffer or 7.5% acetic
acid can be stored protected from light at 4°C for at least three months.
Handling
No toxicity data available. Stock solution contains DMSO and should
be handled with double gloves.
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
34
SYPRO™ Red
Benefits
© Simple, quick staining protocol.
© As sensitive as silver staining (~2ng/band for most proteins)
© Linear range from 2ng to at least 1µg.
© Allows western transfer and immunodetection after staining.
Uses
Nonspecific fluorescent staining of native and denatured proteins in 1D
and 2D SDS-polyacrylamide gels.
Excitation Maximum
Proteins detected with SYPRO Red and Storm imaging.
530nm
Emission Maximum
625nm
Method
© Run gel.
© Dilute SYPRO Red 1:5000 in 7.5% acetic acid (enough to cover

gel). Mix well.
© Place gel and stain in a foil-covered container and shake gently for
30 to 60 minutes.
© For samples with high background, destain in 7.5% acetic acid for
five minutes.
© Rinse briefly in distilled water
Alternative Protocol for Western Transfer

Dilute SYPRO Red 1:5000 in Tris/Glycine/Methanol transfer buffer.
Proceed with staining as above. If necessary, destain in transfer buffer.
35 Protein in Gels
Tips Band sharpness and band density will affect limit of detection. The
following detection limits are for gels that have been stained after
© Ensure glass plates and staining containers are completely free of
electrophoresis (post staining) and are approximate. The PMT voltage
grease and fingerprints. settings are suggested and the actual setting will depend on sample
© Use powder-free gloves to handle gels. concentration.
© Mix fresh staining solution immediately before using.
© Staining in acetic acid significantly impairs immunodetection. For Instrument Setup
western blotting, stain in transfer buffer (sensitivity may be slightly FluorImager 595
diminished). Excitation 514nm
© Staining solution can be reused effectively at least three times. Emission Filter 610RG
© When staining native gels, soak gels in 0.1% SDS (in either 7.5%
Storm
acetic acid or water) for 30 to 60 minutes prior to staining.
(Red Fluorescence)
Sensitivity will be diminished in native gels.
© Methanol can adversely affect staining.
© When staining acidic proteins, increase the concentration of SDS in
Detection Limits
the gel and buffers to 0.2%.
© Phospholipids will not be stained as effectively as other proteins. (ng/band)
FluorImager 2
Storm 3
Binding As for all protein staining systems, dye binding will depend on the
Nonspecific, SDS-dependent binding to proteins. amino acid composition of the proteins. Proteins in native gels will
exhibit more staining variability then proteins saturated with SDS.

Storm ~500-fold
Storage
Stock solution is stable for six months to one year stored at –20°C and
protected from light. Staining reagent diluted in buffer or 7.5% acetic
acid can be stored protected from light at 4°C for at least three months.
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
36
ECF™ Western Blotting Kit
Benefits
© Greater dynamic range of the FluorImager and Storm systems than
film permits visualization and quantitation of weak and dark bands
in the same scan.
screens.
© Allows possibility of direct detection of fluorescein on the
FluorImager system for high target applications.
© Reagent system specifically developed for Molecular Dynamics
Fluorescence scanning systems.
© Dynamic range gives fast quantifiable blots with equivalent
sensitivity to ECL™ westerns.
Uses
Western blotting Western detection of α-fetoprotein in IEF fractions.
ELISA Image courtesy of Paul Cheng, Chinese University of Hong Kong.
Excitation Maximum
ECF substrate 440nm
Excitation
450 or 488nm Fluorescence
510-530nm
Emission Maximum
Fluorescein label 520nm ECF Substrate
Fluorescent
ECF substrate 560nm Product
Alkaline
Phosphate
Method Phosphatase
Group
The following steps summarize the procedure for western blotting:

3° Antibody
1 hr to overnight Electrophoresis
1 to 2 hr Blotting
1 hr Blocking Fluorescein
30 min Washing
1 hr Incubation with primary antibody 2° Antibody
3 min Washing
1° Antibody
1 hr Incubation with fluorescein-linked secondary
antibody Target Protein
30 min Washing (for direct quantitation, the blot can be Solid Support
scanned at this stage)
1 hr Incubation with tertiary antibody (alkaline
phophatase-linked anti-fluorescein)
The secondary antibodies are fluorescently labeled
30 min Washing and can often be detected without further signal
amplification. When further sensitivity is required,
20 min Incubation with ECF substrate and scanning apply the tertiary antibody and develop the blot with
the ECF substrate.
37 Protein on Membranes
© Ensure complete immersion of membrane in all solutions. FluorImager
© Pre-wet PVDF membrane with methanol. Do not allow membrane
Excitation 488nm
to dry out before incubation with ECF substrate. If this happens, Emission Filter 530DF30-fluorescein
wet briefly in methanol and rinse with buffer. 570DF30-ECF
© Use of Amersham Hybond™ PVDF membrane is recommended Storm
over nitrocellulose. (Blue Fluorescence/Chemifluorescence)
© If nitrocellulose must be used, blot must be kept wet. Some signal PMT Voltage 600 to 700
diffusion may result. (Be aware that high PMT voltage can result in membrane background
© If membranes are stored wet, signal will diffuse. Dry blots can be saturation.)
scanned up to several weeks later.
© Blots containing fluorescein should be stored in a dry, dark place at Detection Limits (ECF only)
4°C.
FluorImager (Comparable to Amersham ECL western
© Load 10µl of 1:320 diluted fluorescein-linked anti-mouse-IgG as a blotting)
control for both electrophoresis and scanning (with signal amplifi- Storm (Comparable to Amersham ECL western
cation). For direct fluorescein scanning without signal amplification, blotting)
use a dilution of 1:20.
© Use as large a volume of wash buffer as possible for each wash.
© For direct detection, fluorescein-linked secondary antibody can be
diluted 1:100 instead of the usual 1:600 to extend the linear range. FluorImager 50- to 100-fold
Storm 50- to 100-fold
sterilizing or by rinsing with 0.01M HCl or boiling water to Storage
reduce background resulting from ambient alkaline phosphatase. At 2°C, protected from light
© Incubation with ECF substrate can be extended to 120 minutes for
Reconstituted ECF substrate should be divided into aliquots and frozen
increased sensitivity. for storage longer than two to four weeks.
© Membranes can be stripped and reprobed, if necessary. See protocol
provided with the kit.
Handling
Binding Certain components of the kit are harmful. Use of powder-free gloves
Primary antibody binds specifically to the relevant protein of interest. reagents.
Secondary antibody (conjugated to fluorescein raised against the species
in which the primary was produced) will bind to that primary Vendor
antibody (see diagram overleaf).
Tertiary antibody raised against fluorescein will bind to the fluorescein Amersham Pharmacia Amersham Pharmacia
moiety attached to the secondary antibody. Biotech, Inc., USA Biotech AB, Sweden
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Alkaline phosphatase linked to the tertiary antibody converts ECF
substrate to the fluorescent product, which remains in the region in Amersham Pharmacia www.apbiotech.com
which it is produced. It is believed to adhere to the membrane by Biotech Ltd., England
charge interactions. Tel: +44 1494 544000
38
ECF™ Western Blotting
Reagent Pack
Benefits
© Greater dynamic range than film enables quantitation of both
1 ng
strong and weak bands in the same sample.
© Eliminates the need for darkroom, film and chemiluminescent Dilution series of purified tubulin detected with the ECF
screens. Western Blotting Reagent Pack.
© Optimized for use with Molecular Dynamics Storm and FluorIm-
ager systems.
© Detection protocol and sensitivity comparable to Amersham ECL.
Uses
Western blotting
ELISA
4
Excitation Maximum
Volume x107 (rfu) 3
440nm
2
0.5
Emission Maximum 0.4
0.3
560nm 1 0.2
0.1
0
0 2 4 6 8
0
Method 0 25 50 75 100 125
Tubulin (ng)
The following steps summarize the procedure for western blotting:
Linear range of chemifluorescence on a western blot
1 hr to overnight Electrophoresis
1 to 2 hr Blotting
1 hr Blocking
30 min Washing
Fluorescence
1 hr Incubation with primary antibody Excitation 550–570nm
3 min Washing
ECF
1 hr Incubation with alkaline phosphatase-linked Substrate
secondary antibody Fluorescent

product
30 min Washing
20 min Incubation with ECF substrate and scanning
Alkaline
phosphatase
Phosphate
group
2º antibody
1º antibody
Target protein
Solid
support
Protein detection with the ECF Western Blotting Reagent Pack
39 Protein on Membranes
© To minimize background, block for 2 to 12 hours and increase FluorImager
washes to 15 minutes each. Excitation 488 nm
© Ensure complete immersion of membrane in all solutions.
© Pre-wet PVDF membrane with methanol. Do not allow membrane Storm
to dry out before incubation with ECF substrate. If this happens, (Blue Fluorescence/Chemifluorescence)
wet briefly in methanol and rinse with buffer. PMT Voltage 600 to 700
© Use of Amersham Hybond PVDF membrane is recommended over (Be aware that high PMT voltage can result in membrane background
nitrocellulose. saturation.)
© If nitrocellulose must be used, blot must be kept wet. Some signal
diffusion may result.
Detection Limits
© If membranes are stored wet, signal will diffuse. Dry blots can be
scanned up to several weeks later. FluorImager (Comparable to Amersham ECL western
blotting)
© Use as large a volume of wash buffer as possible for each wash.
Storm (Comparable to Amersham ECL western
© Use of powder-free gloves is important, as the powder fluoresces. blotting)
sterilizing or by rinsing with 0.01M HCl or boiling water to Linear Range of Detection
reduce background resulting from ambient alkaline phosphatase.
FluorImager 50- to 100-fold
© Incubation with ECF substrate can be extended to 120 minutes for Storm 50- to 100-fold
increased sensitivity.
© Membranes can be stripped and reprobed, if necessary. See protocol
provided with the kit. Storage
At 2 to 8°C, protected from light
Binding Reconstituted ECF substrate should be divided into aliquots and frozen
for storage longer than two to four weeks.
Primary antibody binds specifically to the relevant protein of interest.
Alkaline phosphatase linked to the secondary antibody converts ECF
substrate to the fluorescent product, which remains in the region in Handling
which it is produced. Certain components of the kit are harmful. Use of powder-free gloves
reagents (ECF substrate).
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
40
PBXL-3™ Antibodies
Benefits
© Direct red fluorescent detection of westerns on Storm 860.
© Improved quantitation compared to ECF and ECL Plus detection.
Uses
Western blotting
Excitation Maximum
630nm
Emission Maximum
666nm Western detection of ß-tubulin using antimouse-
PBXL-3 conjugate and imaging with Storm 860
Method
© Following addition of PBXL-3 secondary antibody and washing,
carefully lift the blot with clean forceps and briefly drain off excess
buffer. Position in an open plastic bag or sheet protector.* Close
the bag or sheet protector slowly over the wet blot and gently wipe
the top plastic sheet with a Kimwipe®, working out any excess
bubbles. Seal the edges of the bag at this stage if desired.
© Place the blot in the plastic sandwich sample side down onto the
glass. A small puddle of water may be used to form a tight interface
between the plastic and the glass. Work bubbles out by wiping back
of blot and cover with a glass plate to keep flat.
* Use sheet protector, transparency sheets or Kapak® specimen bags for blot development. Stiffer
bags, such as Kapak bags (polyester bag made by 3M Corp.), may result in higher background,
but have the advantage of eliminating wrinkles.
41 Protein in Microplates
© Glove powder fluoresces; use powder-free gloves when handling Storm
blots and preparing to scan. (Red Fluorescence)
© Handle membrane carefully using gloves and forceps at all times to
prevent membrane damage which can result in areas of high
background and spotting. Detection Limits
© For best results, keep blot protected from direct light.
Storm
Western blot (Variable depending on target and
Binding antibodies; 5- to 10-fold less sensitive
than ECF)
Anti-species secondary antibody conjugated to PBXL-3 binds to
primary antibody. Signal is detected by direct excitation of PBXL-3
fluorochrome. Linear Range of Detection
Storm ~ 250-fold
Storage
Product should be stored in the dark at 2 to 8°C. Under these
conditions, the product is stable for at least three months.
Handling
Contains sodium azide. Wear powder-free gloves and suitable protec-
tion at all times.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
42
NanoOrange®
Benefits 100000
© Analyze up to 288 protein samples (in three microplates) simulta-

neously.
Median – Background
© Samples can be read up to six hours after preparation. 10000
© Results not affected by reducing agents and/or nucleic acid

contamination.
© Linear range from 0.5 to 10µg/ml (BSA) 1000
Uses
100
Quantitation of proteins in microplates. 0.01 0.1 1 10 100
BSA (ng/ml)
Excitation Maximum Quantitation of BSA using NanoOrange with FluorImager
475nm
Emission Maximum
575nm (varies slightly for different proteins and high protein
concentrations)
Method
© Dilute 10X diluent (provided) in water.
© Dilute NanoOrange 500-fold in 1X diluent.
© Prepare protein dilutions using NanoOrange solution in glass tubes.
© Heat to 90 to 96°C for ten minutes. Cool completely.
© Pipette samples into microplate.
43 Protein in Microplates
© Mix NanoOrange solution fresh immediately before using. FluorImager
© Assay should be performed in glass tubes, not plastic.
Excitation 488nm
© Allow samples to cool completely before scanning. PMT Voltage 600 to 800
© When scanning on Storm, image quality is improved by using Storm
microplate strips so that the wells sit flat on the scanning surface. (Blue Fluorescence/Chemifluorescence)
© Use clear, flat-bottom, low fluorescence microplates (available from
Nunc or Corning Costar).
© Molecular Dynamics DNA Quantitation Software can be used to Detection Limits
quantitate any sample assayed in microplates with a standard
µg/ml
dilution series. In ImageQuant, report only Object Name, Median,
FluorImager 0.5
and Centroid, and set Background Correction to None.
Storm 1.0
© For the most accurate quantitation using ImageQuant software,
draw an ellipse completely within the walls of one well, and copy it As for all protein staining systems, dye binding will depend on the
to the other wells. amino acid composition and hydrophobic character of the proteins.
Binding Linear Range of Detection

NanoOrange undergoes fluorescent enhancement in a hydrophobic µg/ml
environment. Binding is based on nonspecific hydrophobic interaction FluorImager 0.5 to 10
between the dye, buffer detergents, and protein. Storm 1 to 10
Storage
Store NanoOrange protected from light at –20°C. Can be stored at
room temperature protected from light for up to one week. Store
diluent at room temperature. Store BSA standard at –20°C for long-
term, and at 4°C for short-term use. When stored properly, all reagents
are stable for up to one year.
Handling
Vendor
Eugene, OR 97402-9144
Tel: 541.465.8300
Fax: 541.344.6504
44
Fluorescein
Benefits
© Direct fluorescent detection in DNA and protein applications.
© Improved quantitation in western blotting compared to enzyme-
amplified detection.
Cycle sequencing reaction with
© DNA probes stable for at least six months at –20°C. fluorescein-labeled primer detected
© Offers option for multicolor labeling. with the FluorImager
Uses
Oligonucleotide end-labeling (i.e., PCR primer)
Western blotting
Incorporation of labeled dNTP during PCR
Cycle sequencing using labeled primer
Excitation Maximum
495nm
Emission Maximum
520nm
Method
Refer to manufacturer’s instructions for given fluorescein product. Also,
see Fluorescence 5' Oligolabeling Kit, ECF 3' Oligolabeling and Signal
Amplification Kit and ECF Random Prime Labeling and Signal
Amplification Kit in this guide.
Western blot showing detection with

fluorescein-labeled secondary antibody
imaged on FluorImager
45 Multipurpose Labels
© Use powder-free gloves when handling gels and blots. FluorImager
© Store fluorescein product according to manufacturer’s suggestion, Excitation 488nm
protected from light. Emission Filter 530DF30
PMT Voltage 600-800
Storm
Binding (Blue Fluorescence)
Fluorescein is available covalently conjugated to antibodies, streptavidin,
oligonucleotides and nucleotides.
Detection Limits
FluorImager
DNA label (fmol/band)
Polyacrylamide 1
antibodies)
Storm
DNA label (fmol/band)
Polyacrylamide 30 to 50
antibodies)

FluorImager ~100 to 500-fold
Storage
Store according to manufacturer’s instructions for fluorescein product
Handling
Treat as a potential mutagen.
Vendor
Various
46
Cy™5
Benefits
© Direct fluorescent detection in DNA and protein applications for
Storm 860 system
amplified detection
© DNA probes stable for at least six months at –20°C.
© Offers option for two-color labeling.
Uses
Western blotting.
Cycle sequencing using labeled primers.
Excitation Maximum
649nm
Emission Maximum
670nm
Cycle sequencing reactions with Cy5-
labeled primer imaged with Storm 860.
Method
Refer to manufacturer’s instructions for given Cy5 product.
PCR products amplified with Cy5-dCTP.
Direct fluorescent western blot using Cy5 secondary antibody

conjugate.
© Use powder-free gloves when handling gels and blots. Storm
© Store Cy5-labeled DNA at –20°C, protected from light.
(Red Fluorescence)
© Do not use xylene cyanol or bromophenol blue in sample lanes:
these dyes fluoresce with 635nm excitation. Load tracking dye in an
empty lane to monitor migration and use non-migrating blue Detection Limits
dextran in sample buffer to aid loading.
Storm
© Drying of full format polyacrylamide gels onto filter paper allows DNA label (fmol/band)
easier handling and does not significantly reduce signal-to-noise Polyacrylamide 1
ratio. Western blot (Variable depending on target and
© Using Cy5-labeled primers for sequencing requires cycle sequenc- antibodies)
ing to achieve practical sensitivity.
© Using Cy5-labeled nucleotides may require optimization. A ratio of Linear Range of Detection
10% modified to 90% unmodified dCTP in conjunction with Storm ~100 to 500-fold
AmpliTaq® has been successful.
Storage
Binding
Store according to manufacturer’s instructions for given Cy5 product
Cy5 is available covalently conjugated to antibodies, streptavidin, and
nucleotides, and can be attached to the 5’ terminal of oligonucleotides.
Handling
No toxicity data available. Treat as a potential mutagen.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
48
Cy™3
Benefits
© Direct fluorescent detection in DNA and protein applications
amplified detection
© DNA probes stable for at least six months at –20°C.
Amplification of PCR products in presence of Cy3-dCTP and
© Offers option for multicolor labeling.
scanning with FluorImager 595.
Uses
Western blotting
Excitation Maximum
550nm
Emission Maximum
570nm
Method
Refer to manufacturer’s instructions.
© Use powder-free gloves when handling gels and blots. FluorImager 595
© Store Cy3-labeled DNA at –20°C, protected from light.
Excitation 514nm
© Using Cy3-labeled dNTP may require optimization. The use of PMT Voltage 800 to 900
20% Cy3-dCTP with 80% unmodified dCTP has been successfully
used in PCR amplification with AmpliTaq.
Detection Limits
Binding FluorImager 595

DNA end-label (fmol/band)
Cy3 is available covalently conjugated to antibodies, streptavidin, and Polyacrylamide 5
nucleotides and can be attached to the 5' terminal of oligonucleotides. Western blot (Variable depending on target and
antibodies)

Storm ~100-fold
Storage
Store Cy3-labeled nucleotides and oligonucleotides at –20°C. Store
labeled antibodies in the dark at 2 to 8°C. Product is stable for up to
three months when stored under these conditions.
Handling
Some components of this product may be harmful. Wear powder-free
gloves and suitable protection at all times.
Vendor
Tel: +1 800 526 3593 Tel: +46 18 16 50 00
Tel: +44 1494 544000
50
Additional Fluorochromes
The previous sections of this guide cover the most common fluorescence imaging applications with well-characterized protocols and reagents.
However, with the advent of the Molecular Dynamics fluorescence scanning systems, useful new fluorescent reagents are appearing at an accelerat-
ing pace. This section outlines what is known about these new reagents and their potential uses.
Most absorbance and emission spectral profiles of fluorescent reagents are fairly broad. The fact that the listed absorbance peak of a particular
fluorochrome is not within ± 20nm of the scanner light source doesn’t necessarily mean it won’t be detected by a Molecular Dynamics fluorescence
imaging system. Furthermore, some fluorochromes have a second (or additional) absorbance peak or long “tails” toward shorter wavelengths. For
example, Cy3™ has a second peak of significant absorbance at about 512nm.
For reference, excitation (light source) wavelengths of Molecular Dynamics’ FluorImager and Storm systems are shown below. Detection sensitivity
is usually greatest when the fluorochrome’s absorption maximum correlates closely with the excitation wavelength of the imaging system.
Instrument/Mode Light Source Excitation Wavelength

FluorImager 575 Argon ion laser 488nm
FluorImager SI Argon ion laser 488nm
FluorImager 595 Argon ion laser 488nm and 514nm
Storm Blue Fluorescence/
Chemifluorescence Mode Blue LED 450 ± 30nm
Storm Red Fluorescence Mode Diode laser 635 ± 5nm
Molecular Dynamics posts novel fluorescence imaging methods on the Molecular Dynamics World Wide Web site (http://www.mdyn.com).
Visually interesting images are put on display as the “Image of the Month.” To share new information, images and ideas, call, write, fax or email the
Applications Department at Molecular Dynamics.
Molecular Dynamics toll free 800.743.7782

Applications Department Applications Hotline 800.743.7782, press 5
928 East Arques Avenue fax 408.773.8343
Sunnyvale, CA 94086 USA email applications@mdyn.com
Covalently-linked dyes
These dyes can be used to covalently label proteins or nucleic acids. Some dyes are better suited for different molecules, so check with the vendor
for as much information as possible before starting. The dyes are often available in reactive forms such as isothiocyanates or succinimidyl esters that
can be linked to proteins or oligonucleotides. After labeling, the product can usually be separated from residual-free dye by size exclusion chroma-
tography or gel electrophoresis. Further purification of the labeled product from the starting target compound can be performed by HPLC if
necessary. Nucleic acid probes, PCR primers, single nucleotides and antibodies already conjugated to some of the dyes listed are often available from
the suppliers or custom synthesis service companies.
Fluorochrome absorbancemax (nm) emissionmax (nm) Source

Lucifer Yellow 428 535 Molecular Probes
7-Amino-4-methyl- 349 448 Molecular Probes
coumarine (AMCA) Boehringer-Mannheim
DABCSH 449 494 Lambda Fluorescence
Lambda Green 450-480 510-540 Lambda Fluorescence
NBD-X 446 539 Molecular Probes
R-Phycoerythrin 480, 565 578 Molecular Probes
™
Red 613 480, 565 613 Life Technologies
™
Red 670 480,565 670 Life Technologies
DCM 481 644 Exciton
Nile Red 485 525 Molecular Probes
™
Oregon Green 496 524 Molecular Probes
51
Covalently-linked dyes (continued)

Cy™2 489 506 Amersham Pharmacia Biotech
*Fluorescein, Fluoresceiniso-
thiocynate (FITC) 490 520 Widely available
™
Alpha Red 490 520 Exalpha Corp.
Alexa™ 488 491 515 Molecular Probes
Pyrromethene 556 492 533 Exciton
BODIPY® 493/503 493 503 Molecular Probes
FluorX™ 494 520 Amersham Pharmacia Biotech
FAM™ 495 535 Perkin-Elmer/Applied BioSystems Div.
Rhodol Green 495 525 Molecular Probes
™
Oregon Green 500 503 522 Molecular Probes
BODIPY® FL 503 512 Molecular Probes
Rhodamine 123 505 534 Lambda Fluorescence, Molecular Probes
™
Oregon Green 514 511 530 Molecular Probes
Eosin 517 537 Dupont NEN, Molecular Probes,
Lambda Fluorescence
Carboxyrhodamine (R6G) 519 543 Molecular Probes
TET™ 519 545 Perkin-Elmer/Applied BioSystems Div.
JOE™ 525 557 Perkin-Elmer/Applied BioSystems Div.,
Molecular Probes
HEX™ 529 560 Perkin-Elmer/Applied BioSystems Div.
®
BODIPY 530/550 530 550 Molecular Probes
Alexa 532 532 548 Molecular Probes
Tetramethylrhodamine-
isothiocyanate (TRITC) 547 572 Molecular Probes
*Cy™3 550 570 Amersham Pharmacia Biotech
Rhodamine B 555 580 Molecular Probes
™
TAMRA 555 580 Perkin-Elmer/Applied BioSystems Div.
®
BODIPY TR 592 618 Molecular Probes
Lambda Red 664 600-650 664 Lambda Fluorescence
Nile Blue 690 624 660 Exciton
BODIPY 630/650 630 650 Molecular Probes
*PBXL-3™ 633 666 Amersham Pharmacia Biotech
*Cy™5 649 670 Amersham Pharmacia Biotech
Allo-phycocyanin 650 660 Cyanotech Corp, Molecular Probes
*These dyes are referenced in a previous section and are repeated here for reference.
52
DNA binding dyes/intercalators
DNA binding dyes, some of which are intercalators, bind double-stranded DNA and, to a lesser extent, single-stranded DNA and RNA. With some
of these dyes, binding to DNA substantially increases the intensity of their fluorescence. The dyes can be used to detect DNA in gels. The dimeric
dyes (for example, TOTO-1) are noteworthy for their higher affinity and, in some cases, can be used to prestain the sample for electrophoresis.
Typically, these dyes are less effective as post-stains because they do not penetrate the gel matrix as well as the monomeric forms. PicoGreen is
specifically an “in-solution” stain useful for microplate assays. (Absorbance and emission data are for the dye-nucleic acid complex.)

*PicoGreen 498 520 Molecular Probes
*Vistra Green 490 520 Amersham Pharmacia Biotech
*SYBR Green I 494 520 Molecular Probes, FMC
*SYBR Gold 495 537 Molecular Probes
*SYTO 61 630 645 Molecular Probes
YO-PRO-1 491 509 Molecular Probes
YOYO-1 491 509 Molecular Probes
Acridine Orange 502 526 Molecular Probes
TOTO-1 509 526 Molecular Probes
*Ethidium Bromide 526 605 Widely available
Ethidium Homodimer 528 616 Molecular Probes, Lambda Fluorescence
POPO-3 534 570 Molecular Probes
Propidium Iodide 536 617 Widely available
TO-PRO-3 642 661 Molecular Probes
TOTO-3 642 661 Molecular Probes
RNA and single-stranded DNA stains

These dyes can be used to detect RNA and single-stranded DNA in gels. OliGreen is specifically an “in-solution” stain useful for microplate assays.
(Absorbance and emission data are for the dye-nucleic acid complex.)

OliGreen™ 494 522 Molecular Probes
*SYBR Green II 497 513 Molecular Probes, FMC
*SYBR Gold 495 537 Molecular Probes
Acridine Orange 460 513 Widely available
Protein stains
These are nonspecific protein stains useful for imaging proteins. NanoOrange and CBQCA are “in-solution” stains useful for microplate assays.

*SYPRO Orange 472 570 Molecular Probes
CBQCA 485 530 Molecular Probes
*NanoOrange 475 575 Molecular Probes
*SYPRO Red 530 625 Molecular Probes
53
Enzyme substrates
For Southern and northern blots, alkaline phosphatase enzyme substrates work best. These enzyme substrates are nonfluorescent or weakly fluores-
cent. The product of the enzymatic reaction is highly fluorescent. ECF substrate (AttoPhos) is versatile and effective. Other substrates for alkaline
phosphatase and other enzymes may be useful in microplate assays. Currently, there is at least one commercially available horseradish peroxidase
(HRP) substrate for westerns that is compatible with Molecular Dynamics fluorescence imaging systems.

(substrate for)
Vector Red Unknown Unknown Vector Labs

EnzChek DNase Kit (DNase I) 494 520 Molecular Probes
EnzChek Rnase Kit (Rnase A) 497 513 Molecular Probes
EnzChek Protease Kit (Proteases) 503 512 Molecular Probes
*Vistra ECF Substrate
(alkaline phosphatase) 440 560 Amersham Pharmacia Biotech
Fluorescein diphosphate
(alkaline phosphatase) 490 520 Molecular Probes
Fluorescein digalactropyranoside
(ß-galactosidase) 490 520 Molecular Probes
Fluorescein diglycuronide
(ß-glucuronidase) 490 520 Molecular Probes
Fluorescein di-ß-D-
glucopyranoside (ß-glucosidase) 490 520 Molecular Probes
HNPP/Fast Red TR
(alkaline phosphatase) 550 562 Boehringer-Mannheim
*Dimethylacridinone (DDAO)
phosphate (alkaline phosphatase) 633 655 Molecular Probes
DDAO galactoside
(ß-galactosidase) 633 655 Molecular Probes
DDAO GIcU (ß-glucuronidase) 633 655 Molecular Probes
*ECL Plus Substrate (HRP) 420 460 Amersham Pharmacia Biotech
Tagged enzyme substrates

These enzyme substrates are prelabeled with a fluorescent dye. After the enzymatic reaction, converted and unconverted substrate can be separated
by gel electrophoresis or thin layer chromatography.

(substrate for)
®
FAST CAT Green (chlor-
amphenicol acetyl transferase) 503 512 Molecular Probes
NBD phosphatidylcholine 460 534 Molecular Probes
®
BODIPY phosphatidylcholine
(phospholipase) 503 512 Molecular Probes
™
Pep-Tag
(kinases C and cAMP) 560 590 Promega Corp.
54
pH indicators
These dyes undergo spectral shifts or changes in fluorescence intensity as a function of pH.

Fluorescein (fluoresces at
neutral and alkaline pH) 490 520 Widely available
BCECF (fluoresces at
alkaline pH) 505 531 Molecular Probes
®
SNAFL (in acidic conditions;
spectrum shifts to longer wave-
lengths in alkaline conditions) 514 540 Molecular Probes
Calcium indicators
These dyes change fluorescence characteristics in response to the local Ca2+ concentration.

™
Fura Red
(Ca2+ decreases fluorescence) 458 660 Molecular Probes
™
Calcium Green 505 531 Molecular Probes
Fluo 3 (Ca2+ increases fluorescence) 506 526 Molecular Probes
Lipophilic dyes
These dyes are lipophilic and fluoresce in nonpolar solvents. Primulin is primarily used as an aerosol spray reagent for staining lipids on TLC plates.

Primulin ? ? Sigma
Nile Red (in heptane) 485 525 Molecular Probes
55
Notes
56
© Amersham Pharmacia Biotech [Molecular Dynamics] 1999. All rights reserved. ImageQuant, Storm, PhosphorImager, Personal Densitometer and FluorImager are trademarks of
Molecular Dynamics. Amersham, ECL, Cy, Cy2, Cy3, Cy5, Hybond, FluorX, Surepure, and Vistra Green are trademarks of Amersham Pharmacia Biotech. BODIPY, FAST CAT, Fura Red,
Nano Orange, Oli Green, Oregon Green, Pico Green, POPO-3, SNAFL, SYBR, SYPRO, TO-PRO-3, TOTO-1, TOTO-3, YO-PRO-1, and YO-YO-1 are trademarks or registered trademarks of
Molecular Probes, Inc. Coomassie is a trademark of Imperial Chemical Industries plc. Red 613 and Red 670 are trademarks of Sigma Chemical Company. Alpha Red is a trademark of
Exalpha Corporation. JOE, TET, HEX, and TAMRA are trademarks of Perkin-Elmer Corporation. AllPro and Pep-Tag are trademarks of Promega Corporation. All other brands, products
or services mentioned are the trademarks, servicemarks, registered trademarks or registered servicemarks of their respective holders. *The Polymerase Chain Reaction (PCR) is
covered by patents owned by Roche Molecular Systems and F. Hoffmann-La Roche Ltd. A license to use the PCR process for certain research and development activities accompanies
the purchase of certain reagents from licensed suppliers such as Amersham Pharmacia Biotech and affiliates when used in conjunction with an authorized thermal cycler. All goods
and services are sold subject to the terms and conditions of sale of the company within the Amersham Pharmacia Biotech group which supplies them. A copy of these terms and
conditions is available on request. Product specifications are subject to change without notice. Amersham Pharmacia Biotech UK Limited, Amersham Place, Little Chalfont,
Buckinghamshire, England HP7 9NA; Molecular Dynamics, Inc., 928 E. Arques Avenue, Sunnyvale, CA 94086-4520. Printed in the USA.
For additional
information contact:
Amersham Pharmacia Biotech UK Limited

Amersham Place
Little Chalfont
Buckinghamshire
England HP7 9NA
Amersham Pharmacia Biotech AB

SE-751
84 Uppsala
Sweden
Amersham Pharmacia Biotech Inc.

800 Centennial Avenue
PO Box 1327 Piscataway, NJ 08855
USA
Amersham Pharmacia Biotech Europe GmbH

Munzinger Strasse 9
D-79111 Freiburg
Germany
Molecular Dynamics, Inc.

928 E. Arques Avenue
Sunnyvale, CA 94086-4520, USA
Tel 1 (800) 333-5703
or (408) 773-1222
Fax (408) 773-1493
email: info@mdyn.com
Web: www.mdyn.com
Visit our web site at:

www.mdyn.com
Part No. 0248-264 Rev1 Pub. 7500-9901

Fluorescence Imaging Apllications Guide

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Fluorescence Imaging Apllications Guide

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Fluorescence

Application/Label Vendor FI 575/SI FI 595 Storm 840 Storm 860

The Molecular Dynamics

The Molecular Dynamics Storm system for gel and

2. Photomultiplier Tube (PMT)

Image courtesy of Dr. Velleman, Max-Planck Institute for Molecular

Gel Casting with EtBr

3 Nucleic Acids in Gels

Casting Gel with SYBR Gold

5 Nucleic Acids in Gels

Linear Range of Detection

Casting Gel with SYBR Green I

7 Nucleic Acids in Gels

Linear Range of Detection

© Incubate DNA with 1:5000 dilution of Vistra Green for 15

Casting Gel with Vistra Green

9 Nucleic Acids in Gels

Emission Maximum RNA molecular weight markers stained

11 Nucleic Acids in Gels

Detection Limits RNA

Linear Range of Detection

13 Nucleic Acids in Gels

© SYTO 61 and EtBr may compete for DNA binding.

(4 hr) Optional SurePure purification (only if unlabeled oligos must O = P_ O OH 3'

Total Time 2 hrs. 15 min.

15 Nucleic Acids in Gels

© When scanning a SurePure™ TLC plate on the FluorImager FluorImager (fmol/band)

1 2µl of a 198µl 10,000

17 Nucleic Acids in Microplates

Low Range Standards

19 Nucleic Acids in Microplates

Day 1 Day 2 Day 3 Day 4

2 to 3 hr Electrophoresis 30 min Fixing DNA to 30 min Stringency washes 5 min Scanning

21 Nucleic Acids on Membranes

23 Nucleic Acids on Membranes

Northern blots (having abundant message)

Fluorescein label 495nm

25 Nucleic Acids on Membranes

Linear Range of Detection

© Dilute 1:1000 in Tris/MgCl2 buffer (above)

© For all procedures, including Southerns, westerns, and northerns,

Protein molecular weight markers stained with SYPRO Orange.

© Dilute SYPRO Orange 1:5000 in 7.5% acetic acid (enough to

Alternative Protocol for Western Transfer

© Staining in acetic acid significantly impairs immunodetection. For FluorImager

© Dilute SYPRO Red 1:5000 in 7.5% acetic acid (enough to cover

Alternative Protocol for Western Transfer

Linear Range of Detection

The following steps summarize the procedure for western blotting:

secondary antibody Fluorescent

Protein detection with the ECF Western Blotting Reagent Pack

© Analyze up to 288 protein samples (in three microplates) simulta-

© Results not affected by reducing agents and/or nucleic acid

Excitation Maximum Quantitation of BSA using NanoOrange with FluorImager

Binding Linear Range of Detection

Western blot showing detection with

Linear Range of Detection

PCR products amplified with Cy5-dCTP.

Direct fluorescent western blot using Cy5 secondary antibody

Binding FluorImager 595

Linear Range of Detection

Instrument/Mode Light Source Excitation Wavelength

Molecular Dynamics toll free 800.743.7782

Fluorochrome absorbancemax (nm) emissionmax (nm) Source