Bart's Cookbook--

Kamps's Western Blotting Protocol


Procedure
Recipes
Comments

ECL or autoradiography?

ECL is an appealing technique because it is quick and very sensitive and does not expose the investigator to radioactivity. The use of radioiodinated protein A to detect bound antibodies is however usually a superior technique.

It is much more quantitative, at least if used in conjunction with a Phosphorimager.

It has a lower background. There is less staining of abundant proteins in the sample, and for reasons that aren't clear, protein A shows much less binding to antibodies present in immunoprecipitates than anti-immunoglobulin antibodies. ECL analysis of immunoprecipitates using a second antibody is almost always blighted by strong staining of the antibody bands and by the staining of abundant proteins in the sample.

Blots developed with iodinated protein A can be re-exposed to film to obtain an optimal image. Alternatively, the Phosphorimager scan can be adjusted to give clear definition of bands.

It is almost as fast as ECL. Despite what was indicated in earlier versions of this protocol, identical incubation and washing intervals can be used. Additionally, a successful blot stained with fresh iodinated protein A can often be visualized in 60 minutes with the Phosphorimager, yielding a quantitative, manipulable image.


 Procedure

This procedure was originally optimized by Mark Kamps for analysis with anti-phosphotyrosine antibodies. It is however applicable to almost any form of western blotting with minor modifications.


1) Run an SDS polyacrylamide gel as you would normally.

Before the gel has finished running, be sure that you have started de-gassing the transfer buffer.

Some people soak the gel in transfer buffer for 15 min before assembling the transfer apparatus. Recently, we have been setting up the transfers immediately and things seem to work fine.

We usually use Immobilon membranes for westerns. Although there are suggestions that nitrocellulose may give a lower background than Immoblion in ECL, most people prefer Immobilon even for ECL.

If you are using Immobilon, be sure to wet it first in methanol briefly and then wash away the methanol with transfer buffer.

If you are using a nitrocellulose membrane, soak in transfer buffer prior to assembling the sandwich.




2a) Assemble the transfer folder described below in a large, baking dish containing enough transfer buffer so that all layers are submerged. The orientation of the electrodes is also demonstrated.
(+) POSITIVE ELECTRODE (ANODE)

******************* Plastic cassette
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Scotch-Brite pads
Three Sheets Whatman 3MM
---------------------- Nitrocellulose or Immobilon Filter
---------------------- Polyacrylamide gel
Three Sheets Whatman 3MM
IIIIIIIIIIIIIIIIIIIIIIIIIIIIIII Scotch-Brite pads
******************* Plastic cassette
(-) NEGATIVE ELECTRODE (CATHODE)



Avoid air bubbles between any of the layers.




3) Place the gel/filter sandwich in the holder such that the proteins will migrate from the gel to the membrane, in the direction of the positive (red) electrode (anode).

4) Transfer for 60 min at 60 Volts (or 3600 Volt X Min).

Some very small proteins will pass through the filter if subjected to electrophoresis for this length of time. If that is the case, shorten the electrophoresis. Conversely, some very large proteins do not transfer efficiently under these conditions and their recovery can be increased by longer electrophoresis.

5) Disassemble. At this point you can either rinse the membrane with rinse buffer (or water) and let it dry until you are ready to do the incubation with antibody, or proceed directly.



To block nonspecific binding sites, incubate the membrane for 15 to 30 min in 3% BSA in rinse buffer . This should be filtered prior to use.

In Mark Kamps's original procedure, he used 5% BSA and 1% ovalbumin. 3% BSA appears to work just as well. 1% BSA may also work satisfactorily, but you may be pushing your luck if you use it.

You can reuse the BSA blocking buffer repeatedly.


If you are not using anti-phosphotyrosine antibodies, you can block with BLOTTO. If you are using anti-phosphotyrosine antibodies, you have to use BSA because the background with BLOTTO is very high.


If you are using ECL, OMIT the azide. It interferes with the chemiluminescent chemistry.



6) Incubate the blot with the appropriately diluted antisera for 1 hr at room temperature in 3% BSA in rinse buffer. For affinity purified anti-phosphotyrosine antibodies, we use 2 micrograms/ml. The origninal lab protocol called for a 2 hr incubation, but that seems unnecessary.



7) Wash the filter

2 x 5 min with rinsing buffer

1 x 5 min with NP40 buffer

then 2 x 5 min with rinsing buffer.

With anti-phosphotyrosine antibodies, longer washes reduce the signal, shorter washes increase the background.



8) Incubate the filter for 30 min with 125I-protein A in 3% BSA in rinse buffer (You can re-use your blocking buffer for this). We use ICN's 68038 (approx. 30mCi/mg specific activity). We use 12-14 microCi (approx. 10 microliters) 125I-protein A in a volume of 30-35 ml blocking buffer. We reuse this solution 3 times without noticing a reduction in signal (see #6 in Comments).

9) Wash again as in step 7.

Damp dry on Whatman 3MM, place on another sheet of paper, overlay with saran wrap and expose to film with a screen or use a phosphorimager.

10) Protein bound to the filter can be stained using a solution of 1microliter/ml Pelican india ink, 0.2% Tween-20, in Tris-buffered saline from the shelf (Margie's). Stain for approximately 20 to 60 minutes. Rinse with water for 1 hr. Dry flat. Be careful. Buffers containing Tween 20 can reduce the binding of antiphosphotyrosine antibodies.


Recipes


Transfer buffer:


Glycine 57.6 g
Tris Base 12.0 g
SDS 3.0 g
Methanol 800 ml
H2O to 4 liters

>Na3VO4 370 mg
(only for analysis of phosphotyrosine-
containing proteins).

This solution should be de-gassed !


Rinse buffer:

10 mM Tris-HCl, pH7.4
0.15 M NaCl
0.01% NaN3


NP40 Rinse Solution:

Rinsing buffer plus 0.05% NP40


Comments


1) Reducing the SDS concentration in the transfer buffer by more than 30% inhibits the transfer of some specific proteins from the gel to nitrocellulose.

2) If you've never used an antibody for western blotting before, it is a good idea to dilute it, say 1:200, 1:500, and 1:1000, and compare the signals and backgrounds that you get .

3) Blocking times can be abbreviated if the "BLOTTO" technique is used. This involves blocking with non-fat dry milk and Tween 20. Although this procedure works as well as or better than BSA for most antisera, it results in a much higher background for anti-phosphotyrosine antisera.

4) Protein standards should be run on the same gel if molecular weights are to be determined. All except myosin will transfer and they can be detected by staining with India ink. Transferring the markers has the advantage that the markers themselves are then part of the membrane surface and therefore will shrink and expand along with the samples themselves. Pre-stained markers are particularly useful because they can help you cut your blot or gel and provide visual confirmation that you transferred your proteins in the right direction.

5) Diluted antisera can be reused. We have sometimes re-used the anti-phosphotyrosine antibody 10 times. You can prolong the useful lifetime of the diluted antibodies if you minimize the loss of liquid during handling. A good way to do this is to keep the diluted antibody in the container in which you incubate the blots. This eliminates losses during pouring and pipeting the antibodies.

125I protein A can also be reutilized; however, it should be used only on blots that have been exposed to the same antibody because of the possibility of cross contamination. (i.e. anti-vinculin antibodies that were released into the 125I protein A from the previous blot now binding to the vinculin band on the next blot analyzed with anti-Src antibodies.)


If you want to refer to this technique, cite:

Kamps MP and Sefton BM, (1988) Identification of multiple novel polypeptide substrates of the v-src, v-yes, v-fps, v-ros, and v-erb-B oncogenic tyrosine protein kinases utilizing antisera against phosphotyrosine. Oncogene 2:305-315 . [Abstract of this article]

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