Академический Документы
Профессиональный Документы
Культура Документы
Dot blotting is a method of applying proteins directly onto a membrane. A dissolved sample is pulled through the membrane by either
applying a vacuum, absorption or intrusion; proteins bind to the membrane and the other sample components pass through. The
proteins on the membrane are then available for analysis. This technique can be used either as a qualitative method for rapid screening
of a large number of samples or as a quantitative technique. It is especially useful for testing the suitability of experimental design
parameters.
The following protocol describes a typical procedure for filtering proteins onto an Immobilon-P membrane.
Please review the instructions supplied with your blotting unit for additional information.
* To ensure that the microporous structure of the membrane is not compressed when placed in the blotting unit, it is recommended that
a second sheet of membrane be placed between the filter paper and the primary membrane.
Set Up
Filter
1. Briefly apply the vacuum to remove excess buffer. Please note that it is important that membrane remains wet while loading samples.
2. With the vacuum off, carefully pipette samples into the wells.
3. Apply vacuum to the blotting unit.
4. When all of the samples have filtered through the membrane, turn off the vacuum.
5. Add buffer to each well to wash down the sides; apply vacuum.
6. When all of the wash buffer has filtered through the membrane, turn off the vacuum.
Note: Do not allow the membrane to dry out if analysis of the bound protein requires the native conformation or enzyme activity.
Stack
1. Prepare the membrane the same as above in the Vacuum Filtration Method. (see Set Up Step 1 a c)
2. Assemble stack as follows (from the bottom up):
Place paper towels on work surface (bottom towels should remain dry throughout blotting procedure).
Place dry filter paper (i.e. Whatman 3MM paper) on paper towels.
Place filter paper (prewet with buffer) on dry filter paper.
Place prewet Immobilon-P membrane on wet filter paper.
3. Spot 1 5 mL of sample onto membrane. Sample should wick into membrane. Membrane should be wet enough to absorb sample,
but not so wet that sample spreads across membrane.
4. After sample is absorbed, place membrane on clean filter paper to dry.
References:
1. Protein Blotting Applications Guide, Technical Protocol TP001, 1997, Millipore Corporation, Bedford, Massachusetts, USA.
2. Oprandy, J.J., Olson, J.G., Scott, T.W. (1988). A rapid dot immunoassay for the detection of serum antibodies to Eastern equine
encephalomyelitis and St. Louis encephalitis viruses in sentinel chickens. Am. J. Trop. Med. Hyg., 38 (1), 181-186.
Ordering Information
Immobilon-P Transfer Membranes
Polyvinylidene fluoride (PVDF) for superior western blots
Description
Qty/Pk
Catalog Number
IPVH00010
26 x 26 cm sheet
10
IPVH304F0
20 x 20 cm sheet
10
IPVH20200
15 x 15 cm sheet
10
IPVH15150
10 x 10 cm sheet
10
IPVH10100
9 x 12 cm sheet
10
IPVH09120
7 x 8.4 cm sheet
50
IPVH07850