UAI1 .

2 Immunoblotting and Immunodetection

Immunoblotting (oIten reIerred to as western blotting) is used to identiIy speciIic antigens
recognized by polyclonal or monoclonal antibodies. Protein samples are solubilized,
usually with sodium dodecyl sulIate (SDS) and reducing agents such as dithiothreitol
(DTT) or 2-mercaptoethanol (2-ME). Eollowing solubilization, the material is separated
by SDS-PAGE (UNIT 6.1). The antigens are then electrophoretically transIerred in a tank
(see Basic Protocol 1) or a semidry transIer apparatus (see Alternate Protocol 1) to a
nitrocellulose, PVDE, or nylon membrane, a process that can be monitored by reversible
staining (see Support Protocol 1) or by Ponceau S staining (see Support Protocol 2).
Previously stained gels may also be blotted (see Alternate Protocol 2).
The transIerred proteins are bound to the surIace oI the membrane, providing access Ior
reaction with immunodetection reagents. All remaining binding sites are blocked by
immersing the membrane in a solution containing either a protein or detergent blocking
agent. AIter probing with the primary antibody, the membrane is washed and the
antibody-antigen complexes are identiIied with horseradish peroxidase (HRPO) or alkaline
phosphatase enzymes coupled to the secondary anti-IgG antibody (e.g., goat antirabbit
IgG). The enzymes are attached directly (see Basic Protocol 2) or via an avidin-biotin
bridge (see Alternate Protocol 3) to the secondary antibody. Chromogenic or luminescent
substrates (see Basic Protocol 3 and Alternate Protocol 4) are then used to visualize the
activity. Einally, membranes may be stripped and reprobed (see Support Protocol 3).
NOTE: When handling gels and membranes wear powder-Iree gloves.
BASIC
PRO1OCOL 1
PROTEIN BLOTTING WITH TANK TRANSFER SYSTEMS
In this procedure, blotting is perIormed in a tank oI buIIer with the gel in a vertical
orientation, completely submerged between two large electrode panels. In some systems
up to Iour gels can be transIerred at one time. Eor diIIicult-to-transIer proteins (~100 kDa
or hydrophobic; e.g., myosin), tank blotting is preIerable to semidry systems (see Basic
Protocol 2) because prolonged transIers are possible without buIIer depletion. However,
transIers ~1 hr at high power require cooling using a heat exchanger and a circulating
water bath that can maintain a constant transIer temperature oI 10 to 20C.
Materials
Samples Ior analysis
Protein molecular weight standards (UNIT 6.1), prestained (Sigma or
Bio-Rad) or biotinylated (Vector Labs or Sigma)
TransIer buIIer (see recipe)
Powder-Iree gloves
Scotch-Brite pads (3M) or equivalent sponge
Whatman 3MM Iilter paper or equivalent
TransIer membrane: 0.45-m nitrocellulose (Millipore or Schleicher & Schuell),
PVDE (Millipore Immobilon P), neutral nylon (Pall Biodyne A), or positively
charged nylon (Pall Biodyne B; Bio-Rad Zetabind) membrane
Electroblotting apparatus (EC Apparatus, Bio-Rad, or Amersham Pharmacia
Biotech)
Indelible pen (e.g., Paper-Mate ballpoint) or soIt lead pencil
Additional reagents and equipment Ior gel electrophoresis (UNIT 6.1) and staining
proteins in gels and on membranes (see Support Protocol 1)
NOTE: Deionized, distilled water should be used throughout this protocol.
Contributed by Sean Gallagher, Scott E. Winston, Steven A. Fuller, and 1ohn G.R. Hurrell
Current Protocols in Cell Biologv (1998) 6.2.1-6.2.20
Copyright 1998 by John Wiley & Sons, Inc.
6.2.1
Electrophoresis
and
Immunoblotting
Electrophorese the protein sample
1. Prepare antigenic samples and separate proteins using small or standard-sized one-
or two-dimensional gels (UNIT 6.1). Include prestained or biotinylated protein molecu-
lar weight standards in one or more gel lanes.
The protein markers will transfer to the membrane ana convenientlv inaicate membrane
orientation ana si:es of proteins after immunostaining.
A varietv of gel si:es ana percentages of acrvlamiae can be usea (UNIT 6.1). Most routinelv
usea are either 14 cm 14 cm 0.75mm gels or 8 cm 10 cm 0.75mm minigels.
Acrvlamiae concentrations varv from 5 to 20, but are usuallv in the 10 to 15 range.
Assemble the immunoblot sandwich
2. When electrophoresis is complete, disassemble gel sandwich and remove stacking
gel. Equilibrate gel 30 min at room temperature in transIer buIIer.
Oil from hanas blocks the transfer.
Match the appropriate transfer buffer to the membrane (see Reagents ana Solutions).
Gel equilibration is requirea to prevent a change in the si:e of the gel auring transfer. Anv
shift in gel aimension will result in a blurrea transfer pattern.
3. Assemble transIer sandwich in a tray large enough to hold the plastic transIer cassette.
Eill with transIer buIIer so that cassette is covered.
The transfer cassette shoula be assemblea unaer buffer to minimi:e trapping of air bubbles.
Use Figure 6.2.1 as a guiae to assemblv.
4. On bottom halI oI plastic transIer cassette, place Scotch-Brite pad or sponge, Iollowed
by a sheet oI Iilter paper cut to same size as gel and prewet with transIer buIIer.
plastic
support
filter paper
gel
nitrocellulose
pad
electroblotting
buffer
direction of
protein transfer
anode cathode
+
+
Figure 6.2.1 mmunoblotting with a tank blotting unit. The polyacrylamide gel containing the
protein is laid on a sheet of filter paper. The uncovered side of the gel is overlaid with a sheet of
membrane precut to the size of the gel plus 1 to 2 mm on each edge, then this membrane is overlaid
with another sheet of filter paper. The filter paper containing the gel and membrane is sandwiched
between Scotch-Brite pads. This sandwich is placed in a plastic support, and the entire assembly
is placed in a tank containing transfer buffer. For transfer of negatively charged protein, the
membrane is positioned on the anode side of the gel. For transfer of positively charged protein, the
membrane is placed on the cathode side of the gel. Charged proteins are transferred electrophoreti-
cally from the gel onto the membrane. Transfer is achieved by applying a voltage of 100 V for 1 to
2 hr (with cooling) or 14 V overnight.
Current Protocols in Cell Biology
6.2.2
Immunoblotting
and
Immunodetection
5. Place gel on top oI Iilter paper. The side oI the gel touching the paper arbitrarily
becomes the cathode side oI the gel (i.e., ultimately toward the negative electrode
when positioned in the tank). Remove any air bubbles between gel and Iilter paper
by gently rolling a test tube or glass rod over surIace oI gel.
Anv bubbles between the filter paper, gel, ana membrane will block current flow ana prevent
protein transfer. This problem is inaicatea on the membrane bv sharplv aefinea white areas
aevoia of transferrea protein.
Proteins are usuallv negativelv chargea in transfer buffer ana move towara the positive
anoae. However, some proteins mav be positivelv chargea. An aaaitional membrane placea
on the cathoae siae of the gel will bina these proteins.
6. Prepare transIer membrane. Cut membrane to same size as gel plus 1 to 2 mm on
each edge. Place into distilled water slowly, with one edge at a 45 angle. Equilibrate
10 to 15 min in transIer buIIer.
The water will wick up into the membrane, wetting the entire surface. If it is insertea too
quicklv into the water, air gets trappea ana will appear as white blotches in the membrane.
Protein will not transfer onto these areas.
This wetting proceaure works for nitrocellulose ana nvlon membranes onlv. PJDF mem-
branes are hvarophobic ana will not wet simplv from being placea into aistillea water or
transfer buffer. For these membranes, first immerse 1 to 2 sec in 100 methanol, then
equilibrate 10 to 15 min with transfer buffer. Do not let membrane arv out at anv time. If
this occurs, wet once again with methanol ana transfer buffer as aescribea above.
7. Moisten surIace oI gel with transIer buIIer. Place prewetted membrane directly on
top side oI gel (i.e., anode side) and remove all air bubbles as in step 5.
Poor contact between the gel ana membrane will cause a swirlea pattern of transferrea
proteins on the membrane. Some proteins will transfer as soon as the gel is placea on the
membrane, repositioning the gel or membrane can result in a smearea or aouble image on
the aevelopea blot.
The use of 0.2-m membranes mav improve retention of smaller-molecular-weight pro-
teins.
8. Wet another piece oI Whatman 3MM Iilter paper, place on anode side oI membrane,
and remove all air bubbles. Place another Scotch-Brite pad or sponge on top oI this
Iilter paper.
9. Complete assembly by locking top halI oI the transIer cassette into place (Eig. 6.2.1).
It is important to orient the sanawich so that the membrane faces the anoae (positivelv
chargea) siae of the tank.
1ransfer proteins from gel to membrane
10. Eill tank with transIer buIIer and place transIer cassette containing sandwich into
electroblotting apparatus in correct orientation. Connect leads oI power supply to
corresponding anode and cathode sides oI electroblotting apparatus.
Transfer buffer shoula cover the electroae panels but shoula not touch the base of the
banana plug.
11. Electrophoretically transIer proteins Irom gel to membrane Ior 30 min to 1 hr at 100
V with cooling or overnight at 14 V (constant voltage), in a cold room.
Transfer time is aepenaent on the thickness ana the percent acrvlamiae of the gel, as well
as the si:e of the protein being transferrea. In general, proteins are transferrea within 1 to
6 hr, but high-molecular-weight molecules mav take longer. Overnight transfer at low
voltage is reliable ana convenient. Cooling (at 10 to 20C) is requirea for transfers ~1 hr
at high power. Heat exchanger cooling cores using a circulating water bath are placea into
the transfer unit for cooling.
Current Protocols in Cell Biology
6.2.3
Electrophoresis
and
Immunoblotting
12. Turn oII the power supply and disassemble the apparatus. Remove membrane Irom
blotting apparatus and note orientation by cutting a corner or marking with a soIt lead
pencil or Paper-Mate ballpoint pen.
Manv ballpoint inks come off, but Paper-Mate stavs on the membrane.
Membranes can be ariea ana storea in resealable plastic bags at 4C for 1 vear or longer
at this point. Prior to further processing, ariea PJDF membranes must be placea into a
small amount of 100 methanol to wet the membrane, then in aistillea water to remove
the methanol.
13. Stain gel Ior total protein with Coomassie blue to veriIy transIer eIIiciency. II desired,
stain membrane reversibly to visualize transIerred proteins (see Support Protocol 1),
or irreversibly with Coomassie blue, India ink, naphthol blue, or colloidal gold.
These staining proceaures are incompatible with nvlon membranes.
If membrane shows significant staining on the backsiae, either the gel was heavilv
overloaaea or the membrane has poor protein-binaing capacitv (see Troubleshooting). In
either case, protein-binaing sites on the siae facing the gel are saturatea, allowing protein
to migrate to the other siae of the membrane. Nitrocellulose in particular will show
aiminishea binaing capacitv with age or poor storage conaitions (e.g., high temperature
ana humiaitv). In aaaition, some proteins simplv ao not bina well to a particular matrix.
Bv using several membrane sheets in place of one, the protein can be aetectea as it passes
through each consecutive sheet. This will give an inaication of how efficientlv the membrane
binas to a particular protein.
14. Proceed with immunoprobing and visual detection oI proteins (see Basic Protocols
2 and 3 and Alternate Protocols 3 and 4).
AL1ERAA1E
PRO1OCOL 1
PROTEIN BLOTTING WITH SEMIDRY SYSTEMS
Even and eIIicient transIer oI most proteins is also possible with semidry blotting, a
convenient alternative to tank transIer systems. Instead oI being placed vertically into a
tank Iilled with transIer buIIer, the gel is held horizontally between buIIer-saturated
blotting paper that is in contact with the electrodes (Eig. 6.2.2), greatly reducing the
amount oI buIIer required. The electrodes are close together, giving high Iield strengths
and rapid transIer with a standard electrophoresis power supply. Prolonged transIers (~1
hr) are not recommended; tank blotting (see Basic Protocol 1) should be used Ior proteins
that require long blotting times Ior eIIicient transIer.
Additional Materials (also see Basic Protocol 1)
Six sheets oI Whatman 3MM Iilter paper or equivalent, cut to size oI gel
and saturated with transIer buIIer
Semidry transIer unit (Amersham Pharmacia Biotech, Bio-Rad, or Sartorius)
1. Prepare samples and separate proteins using small or standard-sized one- or two-di-
mensional gels (UNIT 6.1).
Because transfer efficiencv aepenas on manv factors (e.g., gel concentration ana thickness,
protein si:e, shape, ana net charge) results mav varv. Below is a guiaeline for 0.75mm
thick SDS-PAGE gels transferrea bv semiarv blotting.
Percent acrylamide
(resolving gel)
Size range transIerred
(100 eIIiciency)
57 29150 kDa
810 1466 kDa
1315 36 kDa
1820 20 kDa
Current Protocols in Cell Biology
6.2.4
Immunoblotting
and
Immunodetection
2. Prepare transIer membrane (see Basic Protocol 1, step 6).
3. Disassemble gel sandwich. Remove and discard stacking gel.
Equilibration of the separating gel with transfer buffer is not normallv requirea for semiarv
blotting, but it mav improve transfer in some cases.
4. Place three sheets oI Iilter paper saturated with transIer buIIer on the anode (Eig.
6.2.2).
Most transfer units are aesignea so that negativelv chargea proteins move aownwara
towara either a platinum or graphite positive electroae (anoae).
CAPS transfer buffer, pH 10.5 (see recipe for transfer buffer) can be usea in place of the
Tris/glvcine/methanol transfer buffer of Basic Protocol 1. CAPS buffer shoula be usea if
the protein is to be sequencea right on the membrane (Moos, 1992), as glvcine will interfere
with this proceaure.
The filter paper shoula be cut to the exact si:e of the gel. This forces the current to flow
onlv through the gel ana not through overlapping filter paper. Some manufacturers (e.g.,
Amersham Pharmacia Biotech) recommena placing a Mvlar mask on the lower platinum
anoae. With an opening that is slightlv less than the si:e of the gel, the mask forces the
current to flow through the gel ana not the surrounaing electroae area auring transfer.
5. Place equilibrated transIer membrane on top oI Iilter paper stack. Remove all bubbles
between membrane and Iilter paper by rolling a test tube over surIace oI membrane.
Anv bubbles in the filter paper stack or between the filter paper, membrane, ana gel will
block current flow ana prevent protein transfer. This problem is inaicatea on the membrane
bv sharplv aefinea white areas aevoia of transferrea protein.
gel
membrane
buffer-soaked filter paper
transfer
stack
gel
membrane
buffer-soaked filter paper
transfer
stack
cellophane
+ anode
-
cathode
buffer-soaked filter paper
buffer-soaked filter paper
Mylar mask
Figure 6.2.2 mmunoblotting with a semidry transfer unit. Generally, the lower electrode is the
anode, and one gel is transferred at a time. A Mylar mask (optional in some units) is put in place
on the anode. This is followed by three sheets of transfer buffersoaked filter paper, the membrane,
the gel, and finally, three more sheets of buffer-soaked filter paper. To transfer multiple gels, construct
transfer stacks as illustrated, and separate each with a sheet of porous cellophane. For transfer of
negatively charged protein, the membrane is positioned on the anode side of the gel. For transfer
of positively charged protein, the membrane is placed on the cathode side of the gel. Transfer is
achieved by applying a maximum current of 0.8 mA/cm
2
of gel area. For a typical minigel (8 10
cm) and standard-sized gel (14 14 cm), this means 60 and 200 mA, respectively.
Current Protocols in Cell Biology
6.2.5
Electrophoresis
and
Immunoblotting
6. Place gel on top oI membrane. Gently roll a test tube over surIace oI gel to ensure
intimate contact between gel and membrane and to remove any interIering bubbles.
Poor contact between the gel ana membrane will cause a swirlea pattern of transferrea
proteins on the membrane. Some proteins will transfer as soon as the gel is placea on the
membrane, repositioning the gel or membrane can result in a smearea or aouble image on
the aevelopea blot.
7. Complete the transIer stack by putting the three remaining sheets oI Iilter paper on
top oI gel. Roll out bubbles as described above.
Multiple gels can be transferrea using semiarv blotting. Simplv put a sheet of porous
cellophane (Amersham Pharmacia Biotech) or aialvsis membrane (Bio-Raa or Sartorius)
equilibratea with transfer buffer between each transfer stack (Fig. 6.2.2). Transfer effi-
ciencv is aepenaent on the position of the transfer stack in the blotting unit ana for critical
applications transferring one gel at a time is recommenaea. The gel next to the anoae tenas
to be more efficientlv transferrea when blotting more than one gel at a time.
1ransfer proteins from gel to membrane
8. Place top electrode onto transIer stack.
Most units have safetv-interlock features ana can onlv be assemblea one wav. Consult
manufacturers instructions for aetails.
Once assemblea, ao not move the top electroae. This can shift the transfer stack ana move
the gel relative to the membrane. Some transfer will occur as soon as the gel contacts the
membrane, ana anv shifting of the transfer stack after assemblv will aistort the transfer
pattern.
9. CareIully connect high-voltage leads to the power supply (see UNIT 6.1 Ior saIety
precautions). Apply constant current to initiate protein transIer. TransIers oI 1 hr are
generally suIIicient.
In general, ao not exceea 0.8 mA/cm
2
of gel area. For a tvpical minigel (8 10 cm) ana
stanaara-si:ea gel (14 14 cm) this means 60 ana 200 mA, respectivelv.
Monitor the temperature of the transfer unit airectlv above the gel bv touch. The unit shoula
not exceea 45C. If the outsiae of the unit is warm, too much current is being appliea. Note
that units with graphite electroaes are more prone to heating, because graphite has much
more resistance to current flow than platinum or steel electroaes.
10. AIter transIer, turn oII power supply and disassemble unit. Remove membrane Irom
transIer stack, marking orientation as in step 12 oI Basic Protocol 1. Proceed with
staining and immunoprobing (see Basic Protocol 1, steps 13 and 14).
AL1ERAA1E
PRO1OCOL 2
BLOTTING OF STAINED GELS
Gels stained with Coomassie blue R250 can be eIIectively immunoblotted by the
Iollowing procedure, based on Perides et al. (1986) and Dionisi et al. (1995). BrieIly, the
stained gel is soaked in a series oI solutions designed to increase the solubility oI the
proteins aIter staining and Iixation. AIter transIer, the membranes are treated with 45
or 100 methanol to decrease the Coomassie blue bound to the membrane prior to
processing Ior chromogenic development. Eor chemiluminescent development, removal
oI the Coomassie blue is generally not needed.
Materials
Destained gel containing proteins oI interest
25 mM Tris base/192 mM glycine/1 SDS
25 mM Tris base/192 mM glycine/0.1 SDS
Current Protocols in Cell Biology
6.2.6
Immunoblotting
and
Immunodetection
1. Soak destained gel containing proteins oI interest in distilled water Ior 15 min.
2. Equilibrate gel with 25 mM Tris base/192 mM glycine/1 SDS Ior 1 hr with gentle
agitation.
3. TransIer gel to 25 mM Tris base/192 mM glycine/0.1 SDS and equilibrate 30 min
with gentle agitation.
To increase transfer efficiencv of larger proteins, the gel shoula be transferrea to the above
solution with 6 M urea for an aaaitional 30 min.
4. Proceed with transIer (see Basic Protocol 1, steps 2 to 12).
For the most efficient transfer ana binaing to the membrane, the transfer buffer shoula
contain SDS.
5. AIter transIer, soak membranes Ior 10 to 30 min in 45 methanol (nitrocellulose) or
100 methanol (nylon or PVDE) to remove the bound Coomassie blue.
This step is not neeaea if using chemiluminescent reactions or raaiolabelea protein A for
immunoaevelopment. Destaining of the nitrocellulose membrane is enhancea bv aaaing a
small ball of laboratorv tissue to the methanol to absorb the Coomassie blue.
6. Proceed with immunoprobing and visual detection oI proteins (see Basic Protocols
2 and 3 and Alternate Protocols 3 and 4).
SUPPOR1
PRO1OCOL 1
REVERSIBLE STAINING OF TRANSFERRED PROTEINS
To veriIy transIer eIIiciency, nitrocellulose and PVDE membranes can be reversibly
stained. This method will not work on nylon membranes.
Additional Materials (also see Basic Protocol 1)
Ponceau S solution (see recipe)
Additional reagents and equipment Ior photographing membranes
1. Eollowing protein transIer to nitrocellulose or PVDE (see Basic Protocol 1 or
Alternate Protocol 1), place membrane in Ponceau S solution 5 min at room
temperature.
2. Destain 2 min in water. Photograph membrane iI required and mark the molecular-
weight-standard band locations with indelible ink.
3. Completely destain membrane by soaking an additional 10 min in water.
SUPPOR1
PRO1OCOL 2
QUANTITATION OF PROTEIN WITH PONCEAU S
In addition to qualitatively visualizing proteins on membranes aIter blotting, Ponceau S
provides a convenient method Ior quantiIying the amount oI protein in a given lane. By
eluting the dye oII the strip and reading in a spectrophotometer (A
525
), an internal control
value oI protein on a lane is obtained. This value is used to correct Ior any diIIerences in
protein loading Irom lane to lane. Comparison oI the Ponceau S value to the chemilumi-
nescent or chromogenic immunodetection value determined by densitometry provides a
straightIorward correction Ior lane-to-lane variation. This method works best Ior complex
mixtures where the immunodetected protein represents a small proportion oI the total
protein (Klein et al., 1995).
Additional Materials (also see Basic Protocol 1)
Spectrophotometer and 2-ml cuvette
Current Protocols in Cell Biology
6.2.7
Electrophoresis
and
Immunoblotting
1. Eollowing protein transIer to nitrocellulose, PVDE, or nylon (see Basic Protocol 1 or
Alternate Protocol 1), stain membrane, photograph, and destain (see Support Proto-
col 1).
Membranes shoula be aestainea until the backgrouna becomes white.
2. Mark lanes with a soIt pencil and cut lanes into strips.
3. Place each strip into 7 ml oI distilled water Ior 7 min and remove the resulting solution.
II any particulates are visible, centriIuge 30 min at 2000 rpm to remove them.
4. Read A
525
in a 2-ml cuvette.
Anv variation in gel-to-gel sample loaaing ana blotting efficiencv will be reflectea in a
change in A
525
of the sample lanes when comparea to the control. The change in A
525
can be
calibratea to a known amount of protein loaaea on the control lane. This will be a relative
value, however, since the transfer out of the gel ana binaing to the membrane is rarelv 100.
BASIC
PRO1OCOL 2
IMMUNOPROBING WITH DIRECTLY CON1UGATED SECONDARY
ANTIBODY
Immobilized proteins are probed with speciIic antibodies to identiIy and quantitate any
antigens present. The membrane is immersed in blocking buIIer to Iill all protein-binding
sites with a nonreactive protein or detergent. Next, it is placed in a solution containing
the antibody directed against the antigen (primary antibody). The blot is washed and
exposed to an enzyme-antibody conjugate directed against the primary antibody (secon-
dary antibody; e.g., goat anti-rabbit IgG). Antigens are identiIied by chromogenic or
luminescent visualization (see Basic Protocol 3 and Alternate Protocol 4) oI the anti-
gen/primary antibody/secondary antibody/enzyme complex bound to the membrane.
Tween 20 is a common alternative to protein-blocking agents when using nitrocellulose
or PVDE Iilters.
Materials
Membrane with transIerred proteins (see Basic Protocol 1 or Alternate Protocol 1)
Blocking buIIer appropriate Ior membrane and detection protocol (see recipe)
Primary antibody speciIic Ior protein oI interest
TTBS (nitrocellulose or PVDE) or TBS (nylon; see APPENDIX 2A Ior recipes)
Secondary antibody conjugate: horseradish peroxidase (HRPO)or alkaline
phosphatase (AP)anti-Ig conjugate (Cappel, Vector Labs, Kirkegaard & Perry,
or Sigma; dilute as indicated by manuIacturer and store Irozen in 25-l aliquots
until use)
Heat-sealable plastic bag
Powder-Iree gloves
Plastic box
1. Place membrane in heat-sealable plastic bag with 5 ml blocking buIIer and seal bag.
Incubate 30 min to 1 hr at room temperature with agitation on an orbital shaker or
rocking platIorm.
Usuallv 5 ml buffer is sufficient for two to three membranes (14 14cm si:e). If membrane
is to be strippea ana reprobea (see Support Protocol 3), blocking buffer must contain casein
(for AP svstems) or nonfat arv milk.
Plastic incubation travs are often usea in place of heat-sealable bags, ana can be especiallv
useful when processing large numbers of strips in aifferent primarv antiboav solutions.
2. Dilute primary antibody in blocking buIIer.
Primarv antiboav ailution is aeterminea empiricallv but is tvpicallv 1/100 to 1/1000 for a
polvclonal antiboav (Fig. 6.2.3), 1/10 to 1/100 for hvbriaoma supernatants, ana 1/1000
Current Protocols in Cell Biology
6.2.8
Immunoblotting
and
Immunodetection
for murine ascites fluia containing monoclonal antiboaies. Ten- to one-hunarea-fola higher
ailutions can be usea with alkaline phosphatase or luminescencebasea aetection svs-
tems. Both primarv ana seconaarv antiboav solutions can be usea at least twice, but
long-term storage (i.e., ~2 aavs at 4C) is not recommenaea.
3. Open bag and pour out blocking buIIer. Replace with diluted primary antibody and
incubate 30 min to 1 hr at room temperature with constant agitation.
Usuallv 5 ml ailutea primarv antiboav solution is sufficient for two to three membranes
(14 14cm si:e). Incubation time mav varv aepenaing on confugate usea.
When using plastic travs, the primarv ana seconaarv antiboav solution volume shoula be
increasea to 25 to 50 ml. For membrane strips, incubation travs with inaiviaual slots are
recommenaea. Tvpicallv, 0.5 to 1 ml solution/slot is neeaea.
4. Remove membrane Irom plastic bag with gloved hand. Place in plastic box and wash
Iour times by agitating with 200 ml TTBS (nitrocellulose or PVDE) or TBS (nylon),
10 to 15 min each time.
5. Dilute secondary antibody HRPO- or AP-anti-Ig conjugate in blocking buIIer.
Commerciallv available en:vmeconfugatea seconaarv antiboav is usuallv ailutea 1/200
to 1/2000 prior to use (Harlow ana Lane, 1988).
6. Place membrane in new heat-sealable plastic bag, add diluted HRPO- or AP-anti-Ig
conjugate, and incubate 30 min to 1 hr at room temperature with constant agitation.
When using plastic incubation travs, see step 3 annotation for proper antiboav solution
volumes.
7. Remove membrane Irom bag and wash as in step 4. Develop according to appropriate
visualization protocol (see Basic Protocol 3 or Alternate Protocol 4).
Figure 6.2.3 Serial dilution of primary
antibody directed against the 97-kDa
catalytic subunit of the plant plasma
membrane ATPase. Blot was developed with
HRPO-coupled avidin-biotin reagents
according to the second alternate protocol
and visualized with 4-chloro-1-naphthol
(4CN). Note how background improves with
dilution.
Serum
dilution
1
/
5
0
1
/
1
0
0
1
/
2
0
0
1
/
4
0
0
1
/
8
0
0
1
/
1
6
0
0
1
/
3
2
0
0
1
/
6
4
0
0
M
r
(kDa)
200
116
97
66
43
24
18
Current Protocols in Cell Biology
6.2.9
Electrophoresis
and
Immunoblotting
AL1ERAA1E
PRO1OCOL 3
IMMUNOPROBING WITH AVIDIN-BIOTIN COUPLING TO
SECONDARY ANTIBODY
The Iollowing procedure is based on the Vectastain ABC kit Irom Vector Labs (see
SUPPLIERS APPENDIX). It uses an avidin-biotin complex to attach horseradish peroxidase
(HRPO) or alkaline phosphatase (AP) to the biotinylated secondary antibody. Avidin-bio-
tin systems are capable oI extremely high sensitivity due to the multiple reporter enzymes
bound to each secondary antibody. In addition, the detergent Tween 20 is a popular
alternative to protein-blocking agents when using nitrocellulose or PVDE Iilters.
Additional Materials (also see Basic Protocol 2)
Blocking buIIer appropriate Ior membrane and detection protocol (see recipe)
TTBS (nitrocellulose or PVDE) or TBS (neutral or positively charged nylon; see
APPENDIX 2A Ior recipes)
Vectastain ABC (HRPO) or ABC-AP (AP) kit (Vector Labs) containing the
Iollowing: reagent A (avidin), reagent B (biotinylated HRPO or AP), and
biotinylated secondary antibody (request membrane immunodetection protocols
when ordering)
1. Equilibrate membrane in appropriate blocking buIIer in heat-sealed plastic bag with
constant agitation using an orbital shaker or rocking platIorm. Eor nitrocellulose and
PVDE, incubate 30 to 60 min at room temperature. Eor nylon, incubate 2 hr at 37C.
TTBS is well suitea for aviain-biotin svstems. For nvlon, protein-binaing agents are
recommenaea. Because nonfat arv milk contains resiaual biotin, which will interfere with
the immunoassav, it must be usea in the blocking step onlv. If membrane is to be strippea
ana reprobea (see Support Protocol 3), blocking buffer must contain casein (for AP
svstems) or nonfat arv milk.
Plastic incubation travs are often usea in place of heat-sealable bags, ana can be especiallv
useful when processing large numbers of strips in aifferent primarv antiboav solutions.
2. Prepare primary antibody solution in TTBS (nitrocellulose or PVDE) or TBS (nylon).
Dilutions of sera containing primarv antiboav generallv range from 1/100 to 1/100,000.
This aepenas in large part on the sensitivitv of the aetection svstem. With high-sensitivitv
aviain-biotin svstems, ailutions from 1/1000 to 1/100,000 are common. Higher ailutions
can be usea with AP- or luminescence-basea aetection svstems. To aetermine the appro-
priate concentration of the primarv antiboav, a ailution series is easilv performea with
membrane strips. Separate antigens on a preparative gel (i.e., a single large sample well)
ana immunoblot the entire gel. Cut 2- to 4-mm strips bv hana or with a membrane cutter
(Schleicher ana Schuell, Inotech) ana incubate inaiviaual strips in a set of serial ailutions
of primarv antiboav. The correct ailution shoula give low backgrouna ana high specificitv
(Fig. 6.2.3).
3. Open bag, remove blocking buIIer, and add enough primary antibody solution to cover
membrane. Incubate 30 min at room temperature with gentle rocking.
Usuallv 5 ml ailutea primarv antiboav solution is sufficient for two to three membranes
(14 14cm si:e). Incubation time mav varv aepenaing on confugate usea.
When using plastic travs, the primarv ana seconaarv antiboav solution volume shoula be
increasea to 25 to 50 ml. For membrane strips, incubation travs with inaiviaual slots are
recommenaea. Tvpicallv, 0.5 to 1 ml solution/slot is neeaea.
4. Remove membrane Irom bag and place in plastic box. Wash membrane three times
over a 15-min span in TTBS (nitrocellulose or PVDE) or TBS (nylon). Add enough
TTBS or TBS to Iully cover the membrane (e.g., 5 to 10 ml/strip or 25 to 50 ml/whole
membrane).
Current Protocols in Cell Biology
6.2.10
Immunoblotting
and
Immunodetection
5. Prepare biotinylated secondary antibody solution by diluting two drops biotinylated
antibody with 50 to 100 ml TTBS (nitrocellulose or PVDE) or TBS (nylon).
This ailution gives both high sensitivitv ana enough volume to easilv cover a large 14
14cm membrane.
6. TransIer membrane to Iresh plastic bag containing secondary antibody solution.
Incubate 30 min at room temperature with slow rocking, then wash as in step 4.
When using plastic incubation travs, see step 3 annotations for proper antiboav solution
volumes.
7. While membrane is being incubated with secondary antibody, prepare avidin-biotin-
HRPO or -AP complex. Mix two drops Vectastain reagent A and two drops reagent
B into 10 ml TTBS (nitrocellulose or PVDE) or TBS (nylon). Incubate 30 min at
room temperature, then Iurther dilute to 50 ml with TTBS or TBS.
Diluting the A ana B reagents to 50 ml expanas the amount of membrane that can be probea
without greatlv affecting sensitivitv. Soaium a:iae is a peroxiaase inhibitor ana shoula not
be usea as a preservative. Casein, nonfat arv milk, serum, ana some graaes of BSA mav
interfere with the formation of the aviain-biotin complex ana shoula not be usea in the
presence of aviain or biotin reagents (Gillespie ana Huaspeth, 1991, Jector Labs).
8. TransIer membrane to avidin-biotin-enzyme solution. Incubate 30 min at room
temperature with slow rocking, then wash over a 30-min span as in step 4.
9. Develop membrane according to the appropriate visualization protocol (see Basic
Protocol 3 or Alternate Protocol 4).
BASIC
PRO1OCOL 3
VISUALIZATION WITH CHROMOGENIC SUBSTRATES
Bound antigens are typically visualized with chromogenic substrates. The substrates
4CN, DAB/NiCl
2
, and TMB are commonly used with horseradish peroxidase (HRPO)
based immunodetection procedures, while BCIP/NBT is recommended Ior alkaline
phosphatase (AP)based procedures (see Table 6.2.1). AIter incubation with primary and
secondary antibodies, the membrane is placed in the appropriate substrate solution.
Protein bands usually appear within a Iew minutes.
Materials
Membrane with transIerred proteins and probed with antibody-enzyme complex
(see Basic Protocol 2 or Alternate Protocol 3)
TBS (APPENDIX 2A)
Chromogenic visualization solution (Table 6.2.1)
Additional reagents and equipment Ior gel photography
1. II Iinal membrane wash (see Basic Protocol 2, step 7, or see Alternate Protocol 3,
step 8) was perIormed in TTBS, wash membrane 15 min at room temperature in 50
ml TBS.
The Tween 20 in TTBS interferes with 4CN aevelopment (Bferrum et al., 1988).
2. Place membrane into chromogenic visualization solution. Bands should appear in 10
to 30 min.
3. Terminate reaction by washing membrane in distilled water. Air dry and photograph
Ior a permanent record.
Current Protocols in Cell Biology
6.2.11
Electrophoresis
and
Immunoblotting
AL1ERAA1E
PRO1OCOL 4
VISUALIZATION WITH LUMINESCENT SUBSTRATES
Antigens can also be visualized with luminescent substrates. Detection with light oIIers
both speed and enhanced sensitivity over chromogenic and radioisotopic procedures.
AIter the Iinal wash, the blot is immersed in a substrate solution containing luminol Ior
horseradish peroxidase (HRPO) systems or dioxetane phosphate Ior alkaline phosphatase
(AP) systems, sealed in thin plastic wrap, and placed Iirmly against Iilm. Exposures range
Irom a Iew seconds to several hours, although typically strong signals appear within a Iew
seconds or minutes.
TabIe 6.2.1 Chromogenic and Luminescent Visualization Systems
a
System Reagent
b
Reaction/Detection Comments
c
Chromogenic
HRPO-based 4CN Oxidized products Iorm purple
precipitate
Not very sensitive (Tween 20
inhibits reaction); Iades rapidly
upon exposure to light
DAB/NiCl
2
a
Eorms dark brown precipitate More sensitive than 4CN but
potentially carcinogenic; resulting
membrane easily scanned
TMB
e
Eorms dark purple stain More stable, less toxic than
DAB/NiCl
2
; may be somewhat
more sensitive
e
; can be used with all
membrane types; kits available Irom
Kirkegaard & Perry, TSI, Moss, and
Vector Labs
AP-based BCIP/NBT BCIP hydrolysis produces indigo
precipitate aIter oxidation with
NBT; reduced NBT precipitates;
dark blue-gray stain results
More sensitive and reliable than
other AP-precipitating substrates;
note that phosphate inhibits AP
activity
Luminescent
HRPO-based Luminol/H
2
O
2
/
p-iodophenol
Oxidized luminol substrate gives
oII blue light; p-iodophenol
increases light output
Very convenient, sensitive system;
reaction detected within a Iew
seconds to 1 hr
AP-based Substituted 1,2-
dioxetane-phosphates
(e.g., AMPPD, CSPD,
Lumigen-PPD, Lumi-
Phos 530
f
)
Dephosphorylated substrate gives
oII light
Protocol described gives reasonable
sensitivity on all membrane types;
consult instructions oI reagent
manuIacturer Ior maximum sensi-
tivity and minimum background
(see Troubleshooting)
a
Abbreviations: AMPPD or Lumigen-PPD, disodium 3-(4-methoxyspiro1,2-dioxetane-3,2-tricyclo|3.3.1.1
3,7
| decan}-4-yl)phenyl phosphate; AP,
alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; 4CN, 4-chloro-1-napthol; CSPD, AMPPD with substituted chlorine moiety on
adamantine ring; DAB, 3,3-diaminobenzidine; HRPO, horseradish peroxidase; NBT, nitroblue tetrazolium; TMB, 3,3,5,5-tetramethylbenzidine.
b
Recipes and suppliers are listed in Reagents and Solutions except Ior TMP, Ior which use oI a kit is recommended.
c
See Commentary Ior Iurther details.
a
DAB/NiCl
2
can be used without the nickel enhancement, but it is much less sensitive.
e
McKimm-Breschkin (1990) reported that iI nitrocellulose Iilters are Iirst treated with 1 dextran sulIate Ior 10 min in 10 mM citrate-EDTA (pH 5.0),
TMB precipitates onto the membrane with a sensitivity much greater than 4CN or DAB, and equal to or better than that oI BCIP/NBT.
f
Lumi-Phos 530 contains dioxetane phosphate, MgCl
2
, CTAB (cetyltrimethylammonium bromide), and Iluorescent enhancer in a pH 9.6 buIIer.
Current Protocols in Cell Biology
6.2.12
Immunoblotting
and
Immunodetection
Additional Materials (also see Basic Protocol 3)
Luminescent substrate buIIer: 50 mM TrisCl, pH 7.5 (HRPO; APPENDIX 2A) or
dioxetane phosphate substrate buIIer (alkaline phosphatase; see recipe)
Nitro-Block solution (AP reactions only): 5 (v/v) Nitro-Block (Tropix) in
dioxetane phosphate substrate buIIer, prepared just beIore use
Luminescent visualization solution (Table 6.2.1)
Clear plastic wrap
Additional reagents and equipment Ior autoradiography (UNIT 6.3)
NOTE: See Troubleshooting section Ior suggestions concerning optimization oI this
protocol, particularly when employing AP-based systems.
1. Equilibrate membrane in two 15-min washes with 50 ml substrate buIIer.
For blots of whole gels, use 50 ml substrate buffer, for strips, use 5 to 10 ml/strip.
2. For AP reactions using nitrocellulose or PJDF membranes: Incubate 5 min in
Nitro-Block solution, Iollowed by 5 min in substrate buIIer (volumes as in step 1).
Nitro-Block enhances light output from the aioxetane substrate in reactions using AMPPD,
CSPD, or Lumigen-PPD concentrate. It is requirea for nitrocellulose ana recommenaea
for PJDF membranes. It is not neeaea for Lumi-Phos 530, AP reactions on nvlon
membranes, or HRPO-basea reactions on anv tvpe of membrane. Lumi-Phos 530 is not
recommenaea for nitrocellulose membranes.
3. TransIer membrane to visualization solution. Soak 30 sec (HRPO reactions) to 5 min
(AP reactions; volumes as in step 1).
Alternativelv, lav out a square of plastic wrap ana pipet 1 to 2 ml visuali:ation solution
into the miaale. Place membrane on the plastic so that the visuali:ation solution spreaas
out evenlv from eage to eage. Fola wrap back onto membrane, seal, ana proceea to step 5.
4. Remove membrane, drain, and place Iace down on a sheet oI clear plastic wrap. Eold
wrap back onto membrane to Iorm a liquid-tight enclosure.
To ensure an optimal image, onlv one laver of plastic shoula be between the membrane
ana film. Sealable bags are an effective alternative. Moisture must not come in contact with
the X-rav film.
5. In a darkroom, place membrane Iace down onto Iilm.
Do this quicklv ana ao not reposition, a aouble image will be formea if the membrane is
movea while in contact with the film. A blurrea image is usuallv causea bv poor contact
between membrane ana film, use a film cassette that ensures a tight fit.
6. Expose Iilm Ior a Iew seconds to several hours.
Tvpicallv, immunoblots proauce verv strong signals within a few seconas or minutes.
However, weak signals mav require several hours to an overnight exposure. If no image is
aetectea, expose film 30 min to 1 hr, ana if neeaea, overnight (see Troubleshooting).
7. II desired, wash membrane in two 15-min washes oI 50 ml TBS and process Ior
chromogenic development (see Basic Protocol 3).
Chemiluminescent ana chromogenic immunoblotting can be easilv combinea on a single
blot to proviae a permanent visual marker of a known protein. First probe membrane with
the chemiluminescent reactions to recora on film. If stripping ana reprobing is neeaea, then
process bv wetting ana NaOH treatment (see Support Protocol 3). For the last reaction,
use chromogenic aevelopment to proauce a permanent visual recora of the blot. Alterna-
tivelv, once the film recora of the chemiluminescent blot is recoraea, the blot can be rinsea
brieflv with aistillea water ana placea in the appropriate chromogenic solution for
chromogenic aevelopment of the blot. This results in a permanent reference stain on the
blot for comparison to the more easilv scannea ana quantitatea film recora.
Current Protocols in Cell Biology
6.2.13
Electrophoresis
and
Immunoblotting
SUPPOR1
PRO1OCOL 3
STRIPPING AND REUSING MEMBRANES
This stripping procedure works with blotted membranes Irom one- and two-dimensional
gels as well as with proteins blotted Irom previously stained gels (Suck and Krupinska,
1996). Reprobing PVDE membranes that have been developed with chemiluminescent
reagents is simple and straightIorward. All residual antibodies are removed Irom the
membrane by Iirst rewetting it in water and then brieIly treating it with NaOH. Although
repeated reprobing can lead to loss oI signal, up to Iive reprobings generally are Ieasible.
The blot should have been previously blocked with 5 nonIat dry milk prior to treatment.
Materials
0.2 M NaOH
1. Wash blot 5 min in distilled water.
In oraer to effectivelv reprobe the membranes, casein (for AP svstems) or nonfat arv milk
must be usea as the blocking agent. Chromogenic aevelopment leaves a permanent stain
on the membrane that is aifficult to remove, ana shoula not be usea when reprobing. The
stain can interfere with subsequent analvsis if reactive banas from sequential immunostain-
ings are close together.
2. TransIer to 0.2 M NaOH and wash 5 min.
3. Wash blot 5 min in distilled water.
4. Proceed with immunoprobing procedure (see Basic Protocol 2 and Alternate Proto-
col 3).
Casein or nonfat arv milk is recommenaea as blocking agent when reprobing membranes.
REAGENTS AND SOLUTIONS
Use aeioni:ea or aistillea water in all recipes ana protocol steps. For common stock solutions, see
APPEADIX 2A, for suppliers, see SUPPLIERS APPEADIX. For selection of appropriate chromogenic or
luminescent solutions, ana for aefinition of abbreviations, see Table 6.2.1.
Alkaline phosphate substrate buffer
100 mM TrisCl, pH 9.5
100 mM NaCl
5 mM MgCl
2
BCIP/AB1 visualization solution
Mix 33 l NBT stock (100 mg NBT in 2 ml at 70 DME, stored 1 year at 4C)
and 5 ml alkaline phosphate substrate buIIer (see recipe). Add 17 l BCIP stock
(100 mg BCIP in 2 ml oI 100 DME, stored 1 year at 4C) and mix. Stable 1 hr
at room temperature.
Recipe is from Harlow ana Lane (1988). Alternativelv, BCIP/NBT substrates mav be
purchasea from Sigma, Kirkegaara & Perrv, Moss, ana Jector Labs.
Blocking buffer
Colorimetric detection:
For nitrocellulose ana PJDF: 0.1 (v/v) Tween 20 in TBS (TTBS; APPENDIX 2A).
For neutral ana positivelv chargea nvlon: Tris-buIIered saline (TBS; APPENDIX 2A)
containing 10 (w/v) nonIat dry milk. Prepare just beIore use.
TTBS can be storea 1 week at 4C.
Luminescence detection:
For nitrocellulose, PJDF, ana neutral nvlon (e.g., Pall Bioavne A): 0.2 casein (e.g.,
Hammarsten grade or I-Block; Tropix) in TTBS (APPENDIX 2A). Prepare just beIore
use.
continuea
Current Protocols in Cell Biology
6.2.14
Immunoblotting
and
Immunodetection
For positivelv chargea nvlon: 6 (w/v) casein/1 (v/v) polyvinyl pyrrolidone
(PVP) in TTBS (APPENDIX 2A). With constant mixing, add casein and PVP to warm
(65C) TTBS. Stir Ior 5 min. Cool beIore use. Prepare just beIore use.
4CA visualization solution
Mix 20 ml ice-cold methanol with 60 mg 4CN. Separately mix 60 l oI 30 H
2
O
2
with 100 ml TBS (APPENDIX 2A) at room temperature. Rapidly mix the two solutions
and use immediately.
DAB/AiCl
2
visualization solution
5 ml 100 mM TrisCl, pH 7.5 (APPENDIX 2A)
100 l DAB stock (40 mg/ml in H
2
O, stored in 100-l aliquots at 20C)
25 l NiCl
2
stock (80 mg/ml in H
2
O, stored in 100-l aliquots at 20C)
15 l 3 H
2
O
2
Mix just beIore use
CAUTION: Hanale DAB carefullv, wearing gloves ana mask, it is a carcinogen.
Suppliers of peroxiaase substrates are Sigma, Kirkegaara & Perrv, Moss, ana Jector Labs.
Dioxetane phosphate substrate buffer
1 mM MgCl
2
0.1 M diethanolamine
0.02 sodium azide (optional)
Adjust to pH 10 with HCl and use Iresh
Traaitionallv, the AMPPD substrate buffer has been a solution containing 1 mM MgCl
2
ana
50 mM soaium carbonate/bicarbonate, pH 9.6 (Gillespie ana Huaspeth, 1991). The use of
aiethanolamine results in better light output (Tropix Western Light instructions).
Alternativelv, 100 mM TrisCl (pH 9.5)/100 mM NaCl/5 mM MgCl
2
can be usea (Sanahu et
al., 1991).
Dioxetane phosphate visualization solution
Prepare 0.1 mg/ml AMPPD or CSPD (Tropix) or Lumigen-PPD (Lumigen; see
Table 6.2.1) substrate in dioxetane phosphate substrate buIIer (see recipe). Prepare
just beIore use. Lumi-Phos 530 (Boehringer Mannheim or Lumigen) is a ready-to-
use solution and can be applied directly to the membrane.
This concentration of AMPPD substrate (240 M) is the minimum recommenaea bv Tropix
Western Light. Ten-fola lower concentrations can be usea but require longer exposures.
Luminol visualization solution
0.5 ml 10 luminol stock |40 mg luminol (Sigma) in 10 ml DMSO|
0.5 ml 10 p-iodophenol stock |optional; 10 mg (Aldrich) in 10 ml DMSO|
2.5 ml 100 mM TrisCl, pH 7.5 (APPENDIX 2A)
25 l 3 H
2
O
2
H
2
O to 5 ml
Prepare just beIore use
Recipe is from Schneppenheim et al. (1991). Premixea luminol substrate mix (Mast Immu-
nosvstems, Amersham ECL, Du Pont NEN Renaissance, Kirkegaara & Perrv LumiGLO)
mav also be usea. p-ioaophenol is an optional enhancing agent that increases light output.
Luminol ana p-ioaophenol stocks can be storea for 6 months at 20C.
Ponceau S solution
Dissolve 0.5 g Ponceau S in 1 ml glacial acetic acid. Bring to 100 ml with water.
Prepare just beIore use.
Current Protocols in Cell Biology
6.2.15
Electrophoresis
and
Immunoblotting
1ransfer buffer
Add 18.2 g Tris base and 86.5 g glycine to 4 liters oI water. Add 1200 ml methanol
and bring to 6 liters with water. The pH oI the solution is 8.3 to 8.4. Eor use with
PVDE Iilters, decrease methanol concentration to 15; Ior nylon Iilters, omit
methanol altogether.
CAPS transfer buffer can also be usea. Aaa 2.21 g cvclohexvlaminopropane sulfonic acia
(CAPS, free acia), 0.5 g DTT, 150 ml methanol, ana water to 1 liter. Aafust to pH 10.5 with
NaOH ana chill to 4C. For proteins ~60 kDa, reauce methanol content to 1 (Moos, 1992).
COMMENTARY
Background Information
Immunoprecipitation has been widely used
to visualize the antigens recognized by various
antibodies, both polyclonal and monoclonal
(UNIT 7.2). However, there are several problems
inherent with this method, including the re-
quirement Ior radiolabeling oI antigen, co-pre-
cipitation oI tightly associated macromole-
cules, occasional diIIiculty in obtaining pre-
cipitating antibodies, and insolubility oI
various antigens (Talbot et al., 1984).
To circumvent these problems, electroblot-
ting (Towbin et al., 1979)subsequently popu-
larized as western blotting or immunoblotting
(Burnette, 1981)was conceived. Immuno-
blotting is a rapid and sensitive assay Ior the
detection and characterization oI proteins that
works by exploiting the speciIicity inherent in
antigen-antibody recognition. It involves the
solubilization and electrophoretic separation oI
proteins, glycoproteins, or lipopolysaccharides
by SDS-PAGE (UNIT 6.1) or urea-PAGE, Iol-
lowed by quantitative transIer and irreversible
binding to nitrocellulose, PVDE, or nylon. This
technique has been useIul in identiIying spe-
ciIic antigens recognized by polyclonal or
monoclonal antibodies and is highly sensitive
(1 ng oI antigen can be detected).
Electroblotting oI previously stained gels is
a convenient way to visualize and document the
gel prior to immunoblotting. TransIer eIIicien-
cies at all molecular weights will be lower with
Iixed and stained gels. This is particularly true
oI proteins ~50 kDa (Perides et al., 1986). The
additional incubation in 6 M urea will signiIi-
cantly increase transIer eIIiciency oI all pro-
teins and is required Ior proteins ~50 kDa.
Ponceau S staining provides an easy method
Ior calibrating and quantitating the amount oI
material on a nitrocellulose or PVDE blot. An
alternative to this method is to use an internal
protein control with a separate antibody probe,
but these tend to be expensive and time-con-
suming to use. Other applications Ior Ponceau
S calibration include monitoring transIer eIIi-
ciency under varied conditions Ior optimization
oI tank and semidry blotting.
Immunoblotted proteins can be detected by
chromogenic or luminescent assays (see Table
6.2.1 Ior a description oI the reagents available
Ior each system, their reactions, and a compari-
son oI their advantages and disadvantages).
Luminescent detection methods oIIer several
advantages over traditional chromogenic pro-
cedures. In general, luminescent substrates in-
crease the sensitivity oI both HRPO and phos-
phatase systems without the need Ior radioiso-
topes. Substrates Ior the latter have only
recently been applied to protein blotting (see
Gillespie and Hudspeth, 1991; Sandhu et al.,
1991; Bronstein et al., 1992). Luminescent de-
tection can be completed in as little as a Iew
seconds; exposures rarely go more than 1 hr.
Depending on the system, the luminescence
can last Ior 3 days, permitting multiple expo-
sures oI the same blot. Eurthermore, the signal
is detected by Iilm, and varying the exposure
can result in more or less sensitivity. Lumines-
cent blots can be easily erased and reprobed
because the reaction products are soluble and
do not deposit on the membrane (see below).
Compared to chromogenic development, the
luminescent image recorded on Iilm is easier to
photograph and to quantitate by densitometry.
Alkaline phosphatasebased luminescent
protocols that achieve maximum sensitivity
with minimum background can be complex,
and the manuIacturer`s instructions should be
consulted (see Reagents and Solutions). The
procedure described in Alternate Protocol 4
gives reasonable sensitivity on nitrocellulose,
PVDE, and nylon membranes with a minimum
oI steps.
Critical Parameters
Eirst and Ioremost, the antibody being used
should recognize denatured antigen. Nonspe-
ciIic binding oI antibodies can occur, so control
antigens and antibodies should always be run
in parallel. Time oI transIer and primary anti-
Current Protocols in Cell Biology
6.2.16
Immunoblotting
and
Immunodetection
body and conjugate dilutions should always be
optimized.
A variety oI agents are currently used to
block binding sites on the membrane aIter blot-
ting (Harlow and Lane, 1988). These include
Tween 20, PVP, nonIat dry milk, casein, BSA,
and serum. A 0.1 (v/v) solution oI Tween 20
in TBS (TTBS), a convenient alternative to
protein-based blocking agents, is recom-
mended Ior chromogenic development oI ni-
trocellulose and PVDE membranes (Blake et
al., 1984). In contrast to dry milk/TBS blocking
solution (BLOTTO), TTBS is stable and has a
long shelI liIe at 4C. Eurthermore, TTBS gen-
erally produces a clean background and permits
subsequent staining with India ink. However,
even with the application oI such standard
blocking procedures as 5 to 10 milk protein
or 0.05 to 0.1 Tween 20, background can
still be a signiIicant problem. II this happens,
using a blocking protein (e.g., goat, horse, or
rabbit normal serum) Irom the same species as
the primary antibody can reduce the back-
ground, presumably by reducing cross-reactiv-
ity between the primary antibodies and the
blocking agent. Combinations oI blocking
agents can also be eIIective. Thus, 0.1 human
serum albumin (HSA) and 0.05 Tween 20 in
TBS is recommended when probing Immo-
bilon-P membranes with human serum (Craig
et al., 1993). However, this can also lead to
overall loss oI antigen signal, requiring a ten-
Iold increase in the primary antibody (serum)
concentration to achieve an adequate back-
ground Iree antigen signal.
When using chemiluminescent detection Ior
immunoblotting, high background Irequently
occurs, particularly Ior strong signals (Pampori
et al., 1995). Several methods are available Ior
reducing the background Irom chemilumines-
cent reactions. These include changing the type
and concentration oI blocking agents (see
above), optimizing antibody concentrations,
letting the reaction proceed Ior several minutes
beIore exposing to Iilm, or simply limiting the
exposure time oI the Iilm on the blot. These
procedures are not always successIul, however,
and can lead to inconsistent results. An alterna-
tive approach is to reduce the concentration oI
reagents ten-Iold. This eIIectively removes the
background and has a number oI advantages
which include lower cost, increased signal-to-
noise ratio, and reduced detection oI cross-re-
acting species.
Two types oI nylon membrane are used Ior
western transIerneutral (e.g., Pall Biodyne
A) and positively charged (e.g., Pall Biodyne
B). Although the positively charged mem-
branes have very good protein-binding charac-
teristics, they tend to give a higher background.
These membranes remain positively charged
Irom pH 3 to pH 10. Neutral nylon membranes
are also charged, having a mix oI amino and
carboxyl groups that give an isoelectric point
oI 6.5. Because oI their high binding capacity,
positively charged membranes are popular Ior
protein applications using luminescence.
Nylon membranes require more stringent
blocking steps. Here 10 nonIat dry milk in
TBS is recommended Ior chromogenic devel-
opment. During luminescence development,
however, background is a more signiIicant
problem. Compared to dry milk, puriIied casein
has minimal endogenous alkaline phosphatase
activity (AP activity leads to high background)
and is thereIore recommended as a blocking
agent Ior nitrocellulose, PVDE, and nylon
membranes. Positively charged nylon requires
much more stringent blocking with 6 (w/v)
casein and 1 (v/v) polyvinylpyrrolidone
(PVP). Because nonIat dry milk and casein may
contain biotin that will interIere with avidin-
biotin reactions, subsequent steps are done
without protein-blocking agents when using
these systems. II background is a problem,
highly puriIied casein (0.2 to 6) added to
the antibody incubation buIIers may help.
II reprobing is desired, blots can be air dried
and stored at 4C Ior 3 months aIter chemilu-
minescence detection. AIter drying, store in a
sealed Ireezer bag until use. Repeated probing
will lead to a gradual loss oI signal and in-
creased background. However, this will depend
in part on the properties oI the sample.
II the primary procedure is problematic due
to loss oI sensitivity or an increase in the back-
ground, then two possible alternative proce-
dures Ior stripping membranes are recom-
mended. The Iirst uses 2-mercaptoethanol and
SDS (KauImann et al., 1987; TesIaigzi et al.,
1994). BrieIly, the membranes are incubated in
2 SDS/100 mM TrisCl, pH 7.4/100 mM
2-mercaptoethanol Ior 30 min at 70C, eIIec-
tively removing primary and secondary anti-
bodies. As with the primary procedure recom-
mended above, the repeated probing should be
done with caution due to the potential loss oI
detection signal, and 5 nonIat dry milk is
required as a blocking agent. The milk blocking
agent Iacilitates antibody removal Irom the blot
(KauImann et al., 1987). The second uses
guanidineHCl. Eor nylon and PVDE mem-
branes (do not use with nitrocellulose), incu-
bate the immunoblot in 7 M guanidineHCl Ior
Current Protocols in Cell Biology
6.2.17
Electrophoresis
and
Immunoblotting
10 min at room temperature. (The short wash
time is critical, as guanidineHCl is a very
strong denaturant, so do not leave the Iilter in
this solution ~15 min.) Pour oII excess guani-
dineHCl and then rinse the membrane several
times in 1 TTBS. Reblock the membrane and
proceed with the standard immunoblotting pro-
cedure. Membranes stripped using this proce-
dure can generally be reused three or Iour times.
Troubleshooting
There are several problems associated with
immunoblotting. The antigen is solubilized and
electrophoresed in the presence oI denaturing
agents (e.g., SDS or urea), and some antibodies
may not recognize the denatured Iorm oI the
antigen transIerred to the membrane. The re-
sults observed may be entirely dependent on
the denaturation and transIer system used. Eor
example, zwitterionic detergents have been
shown to restore the antigenicity oI outer mem-
brane proteins in immunoblotting (Mandrell
and Zollinger, 1984). Gel electrophoresis under
nondenaturing conditions can also be per-
Iormed.
Other potential problems include high back-
ground, nonspeciIic or weak cross-reactivity oI
antibodies, poor protein transIer or membrane
binding eIIiciency, and insuIIicient sensitivity.
Eor an extensive survey and discussion oI im-
munoblotting problems and artiIacts, see Bjer-
rum et al. (1988).
II no transIer oI protein has occurred, check
the power supply and electroblot apparatus to
make sure that the proper electrical connections
were made and that power was delivered during
transIer. In addition, check that the correct ori-
entation oI Iilter and gel relative to the anode
and cathode electrodes was used.
II the transIer eIIiciency using the tank sys-
tem appears to be low, increase the transIer time
or power. Cooling (using the unit`s built-in
cooling cores) is generally required Ior trans-
Iers ~1 hr. At no time should the buIIer tem-
perature go above 45C. Prolonged transIers
(~1 hr) are not possible in semidry transIer units
due to rapid buIIer depletion.
Alternatively, the transIer buIIer can be
modiIied to increase eIIiciency. Adding SDS at
a concentration oI 0.1 to the transIer buIIer
improves the transIer oI all proteins out oI the
gel, particularly those above 60 to 90 kD in size.
Lowering the concentration oI methanol will
also improve the recovery oI proteins Irom the
gel. These procedures are tradeoIIs. Methanol
improves the binding oI proteins to PVDE and
nitrocellulose, but at the same time hinders
transIer. With SDS present, transIer eIIiciency
is improved, but the SDS can interIere with
protein binding to the membrane. Nylon and
PVDE membranes are particularly sensitive to
SDS interIerence. II needed, 0.01 to 0.02
SDS may be used in PVDE membrane transIer
buIIers (Millipore, 1990). SDS and methanol
should not be used in the transIer buIIer Ior
nylon (Peluso and Rosenberg, 1987).
Gel cross-linking and thickness also have a
proIound eIIect on the transIer eIIiciency. In
general, 0.5- to 0.75-mm-thick gels will trans-
Ier much more eIIiciently than thicker gels
(e.g., 1.5 mm thick). Gels with a higher acryl-
amide percentage will also transIer less eIIi-
ciently. Proteins can be particularly diIIicult to
transIer Irom gradient gels, and a combination
oI longer transIer times, thin gels, and the
addition oI SDS to the transIer buIIer may be
needed.
II the protein bands are diIIuse, check the
transIer cassette. The gel must be held Iirmly
against the membrane during transIer. II the
transIer sandwich is loose in the cassette, add
another thin sponge or more blotter paper to
both sides.
Occasionally, a grid pattern will be apparent
on the membrane aIter tank transIer. This is
caused by having either the gel or the membrane
too close to the sides oI the cassette. Correct
this by adding more layers oI Iilter paper to
diIIuse the current Ilowing through the gel and
membrane. Use a thinner sponge and more
Iilter paper iI necessary.
II air bubbles are trapped between the Iilter
and the gel, they will appear as clear white areas
on the Iilter aIter blotting and staining. Take
extra care to make sure that all bubbles are
removed.
InsuIIicient blocking or nonspeciIic binding
oI the primary or secondary antibody will cause
a high background stain. A control using pre-
immune serum or only the secondary antibody
will determine iI these problems are due to the
primary antibody. Try switching to another
blocking agent; protein blocking agents may
weakly cross-react. Lowering the concentra-
tion oI primary antibody should decrease back-
ground and improve speciIicity (Eig. 6.2.3).
Due to the nature oI light and the method oI
detection, certain precautions are warranted
when using luminescent visualization (e.g.,
Harper and Murphy, 1991). Very strong signals
can overshadow nearby weaker signals on the
membrane. Because light will pipe through the
membrane and the surrounding plastic wrap,
overexposure will produce a broad diIIuse im-
Current Protocols in Cell Biology
6.2.18
Immunoblotting
and
Immunodetection
age on the Iilm. The signal can also saturate the
Iilm, exposing the Iilm to a point whereby
increased exposure will not cause a linear in-
crease in the density oI the image on the Iilm.
With the alkaline phosphatase substrate
AMPPD, nitrocellulose, PVDE, and nylon
membranes require 2, 4, and 8 to 12 hr, respec-
tively, to reach maximum light emission. In
addition, PVDE is reported to give a stronger
signal than nitrocellulose (Tropix Western
Light instructions). Positively charged nylon
requires special blocking procedures to mini-
mize background (Gillespie and Hudspeth,
1991). These procedures include using a block-
ing and primary antibody solution containing
6 casein, 1 polyvinylpyrrolidone-40 (PVP-
40), 3 mM NaN
3
, 10 mM EDTA, and PBS, pH
6.8. Prior to use, the casein must be heated to
65C to reduce alkaline phosphatase activity in
the casein itselI. In addition, maximum sensi-
tivity has been observed when Iree biotin or
biotinylated proteins are removed by pretreat-
ing the casein with avidin-agarose (Sigma).
Anticipated Results
Immunoblotting should result in the detec-
tion oI one or more bands. Although antibodies
directed against a single protein should produce
a single band, degradation oI the sample (e.g.,
via endogenous proteolytic activity) may cause
visualization oI multiple bands oI slightly diI-
Ierent size. Multimers will also Iorm spontane-
ously, causing higher-molecular-weight bands
on the blot. II simultaneously testing multiple
antibodies directed against a complex protein
mixture (e.g., using patient sera against SDS-
PAGE-separated viral proteins in AIDS west-
ern blot test), multiple bands will be visualized.
Typically, picogram to nanogram sensitivities
are common in protein blotting and immunode-
tection procedures.
Time Considerations
The entire immunoblotting procedure can
be completed in 1 to 2 days, depending on
transIer time and type oI gel. Gel electrophore-
sis requires 4 to 6 hr on a regular gel and 1 hr
on a minigel. TransIer time can be 1 hr (high-
power transIer) to overnight. Blocking, conju-
gate incubation, and washing each take 30 min
to 1 hr. Einally, substrate incubation requires 10
to 30 min (chromogen) and a Iew seconds to
several hours (luminescence).
Literature Cited
Bjerrum, O.J., Larsen, K.P., and Heegaard, N.H.H.
1988. NonspeciIic binding and artiIacts-speci-
Iicity problems and troubleshooting with an atlas
oI immunoblotting artiIacts. In CRC Handbook
oI Immunoblotting oI Proteins, Vol. I: Technical
Descriptions (O.J. Bjerrum and N.H.H.
Heegaard, eds.) pp. 227-254. CRC Press, Boca
Raton, Ela.
Blake, M.S., Johnston, K.H., Russell-Jones, G.J.,
and Gotschlich, E.C. 1984. A rapid, sensitive
method Ior detection oI alkaline phosphatase
conjugated anti-antibody on Western blots. Anal.
Biochem. 136:175-179.
Bronstein, I., Voyta, J.C., Murphy, O.J., Bresnick,
L., and Kricka, L.J. 1992. Improved chemilumi-
nescent Western blotting procedure. BioTech-
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Burnette, W.N. 1981. Western blotting: Electro-
phoretic transIer oI proteins Irom sodium do-
decyl sulIate-polyacrylamide gels to unmodiIied
nitrocellulose and radiographic detection with
antibody and radioiodinated protein A. Anal.
Biochem. 112:195-203.
Craig, W.Y., Poulin, S.E., Collins, M.E., Ledue, T.B.,
and Ritchie, R.E. 1993. Background staining in
immunoblot assays. Reduction oI signal caused
by cross-reactivity with blocking agents. J. Im-
munol. Methoas 158:67-76.
Dionisi, H.M., Checa, S.K., and Viale, A.M. 1995.
Protein immunoblotting oI stained gels.
BioTechniques 19:348-350.
Gillespie, P.G. and Hudspeth, A.J. 1991. Chemilu-
minescence detection oI proteins Irom single
cells. Proc. Natl. Acaa. Sci. U.S.A. 88:2563-
2567.
Harlow, E. and Lane, D. 1988. Immunoblotting. In
Antibodies: A Laboratory Manual, pp. 471-510.
CSH Laboratory, Cold Spring Harbor, N.Y.
Harper, D.R. and Murphy, G. 1991. NonuniIorm
variation in band pattern with luminol/horserad-
ish peroxidase Western blotting. Anal. Biochem.
192:59-63.
KauImann, S.H., Ewing, C.M., and Shaper, J.H.
1987. The erasable Western blot. Anal. Biochem.
161:89-95.
Klein, D., Kern, R.M., and Sokol, R.Z. 1995. A
method Ior quantiIication and correction oI pro-
teins aIter transIer to immobilization mem-
branes. Biochem. Mol. Biol. Int. 36:59-66.
Mandrell, R.E. and Zollinger, W.D. 1984. Use oI
zwitterionic detergent Ior the restoration oI anti-
body-binding capacity oI electroblotted menin-
gococcal outer membrane proteins. J. Immunol.
Methoas 67:1-11.
McKimm-Breschkin, J.L. 1990. The use oI tetra-
methylbenzidine Ior solid phase immunoassays.
J. Immunological Methoas 135:277-280.
Millipore. 1990. Protein blotting protocols Ior Im-
mobilon-P transIer membrane. BedIord, Mass.
Current Protocols in Cell Biology
6.2.19
Electrophoresis
and
Immunoblotting
Moos, M. 1992. Isolation oI proteins Ior microse-
quence analysis In Current Protocols in Immu-
nology (J.E. Coligan, A.M. Kruisbeek, D.H.
Margulies, E.M. Shevach, and W. Strober, eds.)
pp. 8.7.1-8.7.12. Greene Publishing Associates
and John Wiley & Sons, New York.
Pampori, N.A., Pampori, M.K., and Shapiro, B.H.
1995. Dilution oI the chemiluminescence re-
agents reduces the background noise on Western
blots. BioTechniques 18:588-590.
Peluso, R.W. and Rosenberg, G.H. 1987. Quantita-
tive electrotransIer oI proteins Irom sodium do-
decyl sulIate polyacrylamide gels onto positively
charged nylon membranes. Anal. Biochem.
162:389-398.
Perides, G., Plagens, U., and Traub, P. 1986. Protein
transIer Irom Iixed, stained, and dried polyacry-
lamide gels and immunoblot with protein A
gold. Anal. Biochem. 152:94-99.
Sandhu, G.S., EckloII, B.W., and Kline, B.C. 1991.
Chemiluminescent substrates increase sensitiv-
ity oI antigen detection in Western blots. Bio-
Techniques 11:14-16.
Schneppenheim, R., Budde, U., Dahlmann, N. and
Rautenberg, P. 1991. Luminographya new,
highly sensitive visualization method Ior electro-
phoresis. Electrophoresis 12:367-372.
Suck, R.W.L. and Krupinska, K. 1996. Repeated
probing oI Western blots obtained Irom Coomas-
sie Brilliant Bluestained or unstained polyacry-
lamide gels. BioTechniques 21:418-422.
Talbot, P.V., Knobler, R.L., and Buchmeier, M.
1984. Western and dot immunoblotting analysis
oI viral antigens and antibodies: Application to
murine hepatitis virus. J. Immunol. Methoas
73:177-188.
TesIaigzi, J., Smith-Harrison, W., and Carlson, D.M.
1994. A simple method Ior reusing western blots
on PVDE membranes. BioTechniques 17:268-
269.
Towbin, H., Staehelin, T., and Gordon, J. 1979.
Electrophoretic transIer oI proteins Irom
polyacrylamide gels to nitrocellulose sheets:
Procedure and some applications. Proc. Natl.
Acaa. Sci. U.S.A. 76:4350-4354.
Key References
Bjerrum, O.J. and SchaIer-Nielsen, C. 1986. BuIIer
systems and transIer parameters Ior semidry
electroblotting with a horizontal apparatus. In
Electrophoresis `86 (M.J. Dunn, ed.) pp. 315-
327. VCH Publishers, DeerIield Beach, Ela.
Describes the semiarv blotting svstem.
Gillespie and Hudspeth, 1991. See above.
Describes alkaline phosphataseluminescent aetec-
tion methoas.
Harlow and Lane, 1988. See above.
Details alternative aetection methoas.
Salinovich, O. and Montelaro, R.C. 1986. Revers-
ible staining and peptide mapping oI proteins
transIerred to nitrocellulose aIter separation by
sodium dodecyl sulIate-polyacrylamide gel elec-
trophoresis. Anal. Biochem. 156:341-347.
Describes the use of Ponceau S staining for im-
munoblotting.
Schneppenheim et al., 1991. See above.
Details peroxiaase-basea luminescent aetection
methoas.
Contributed by Sean R. Gallagher
Motorola Corporation
Tempe, Arizona
Scott E. Winston and Steven A.
Euller (tank transIer systems)
Univax Biologics
Rockville, Maryland
John G.R. Hurrell (tank transIer systems;
reversible staining oI proteins)
Boehringer Mannheim Biochemicals
Indianapolis, Indiana
Current Protocols in Cell Biology
6.2.20
Immunoblotting
and
Immunodetection