WESTBLOT� 7/28/98

WESTERN BLOTTING PROTOCOL

A) RUNNING GELS AND TRANSFERRING:

1.� Run minigels (one gel per Bio-Rad mini-protean transfer cassette) or full size gels (7 lane max per cassette).� Transfer to nitrocellulose or PVDF paper in Towbin buffer (see below); use forceps (NOT FINGERS) to hold the membrane at all times.� No special procedure to wet nitrocellulose, but for PVDF wet briefly in MeOH, soak 5 minutes in deionized water, and 15 minutes in towbin buffer.� On white side of transfer cassette, place wet Scotchgard, one peice of wet filter paper (Whatman), the membrane, the gel, two peices of wet filter paper and another scotchgard.� Squeeze bubbles out by splashing buffer and rolling with a pipette after each addition.� Bubbles are very bad.� Close cassette, place in transfer apparatus (make sure poles are lined up (run to red)).�

2.� Add ice and stir bar, place on stir plate and for IR or IRS-1 etc in 7.5% gel, transfer for 1.5 hours at 120 V constant voltage.� Change ice pack 1/2 way through transfer.� Smaller proteins can be transferred at 50 mA overnight or 100 mA for several hours.

3.� Remove gel, membrane, filters from cassette.� Rinse membrane briefly in deionized water, and then place in block buffer at 4 C overnight.� Gel, filter, etc. get trashed.

B)� IMMUNOBLOT TRANSFERRED PROTEINS

4.� After blocking overnight, incubate the membrane in enough antibody solution to cover it and let it move around on the room temperature rocker for two hours.� Antibody solutions are made up in block buffer.� If you are going to use ECL, you should use block buffer WITHOUT azide for all steps.

**NOTE:� MANY USED ANTIBODY SOLUTIONS CAN BE SAVED FOR RE-USE SEVERAL TIMES (some cannot however, so check with someone who knows).

5.� Wash 4 times five minutes with wash buffer.

6.� Reblock the membrane for 1 hour on room temperature rocker in block solution.

7.� Pour off block solution and add for 1 hour on room temperature rocker:

a.� ¹²⁵I-protein A (2 uCi/10 ml) in block buffer OR

b.� HRP conjugated secondary antibody (e.g HRP Goat arabbit or anti-mouse) (Affinity purified from Cappel); 1:5000 in block buffer (ECL protocol).

8.� Pour off solution (¹²⁵I-protein A solutions can be re-used several times for all but the most sensistive of assays; secondary antibody solutions get somewhat weak quickly).� Wash membrane 4+ times 15-20 minutes with wash buffer.

9.� Detect bound antibodies:

a.� (¹²⁵I-protein A protocol)� Dry membrane on bench on filter paper, expose to autoradiography wrapped in saran wrap (else membrane may stick to film and be ruined).� You usually need a screen at -70 C overnight to see a western.� OR

b.� (ECL protocol)� Using forceps, remove the excess buffer from the membrane by touching the edge to a filter paper.� Place membrane in ECL reagent* (5 ml soln A + 5 ml soln B in a blotting box) and incubate with occasional agitation for 30-60 seconds.� Blot the edge of the membrane against filter paper to remove excess liquid and quickly wrap the membrane in plastic wrap and expose (no screen required).� First exposure of 1 minute is a good start.� Vary time as needed, but remember that the solution decays with time....

*NOTES on ECL reagent:

Since once soln A and Soln B are mixed, they become unstable, take care to avoid cross-contamination:� change pipettes, do not exchange bottle caps, etc.� This also means that you shouldn't keep the mixed solution around for more than a few minutes before use.� Reagents are light sensitive.

OTHER NOTES:� ECL works especially well on lysates, since the secondary antibody can recognize the heavy and light chains of the antibody from immunoprecipitations, giving you background.� For some antibodies, the ¹²⁵I-protein A will also recognize the IP antibody on the nitrocellulose. In these cases, it is appropriate to do the IP with antibody covalently coupled to protein A (see DMP coupling method in Harlow and Lane book "Antibodies").

C)� REAGENTS AND SOLUTIONS

Towbin (Transfer) Buffer:� 4 liters

25 mM Tris � 12.1 g Tris base

192 mM Glycine� 57.6 g Glycine

20% MeOH� 800 ml MeOH

0.02% SDS�� 8 ml 10% SDS

Wash Solution� 2 liters

20 mM Tris 7.4� 40 ml 1 M Tris 7.4

150 mM NaCl� 60 ml 5 M NaCl

0.01% Tween-20� 200 ul Tween-20

Block Solution� 500 mls

Wash solution containing:

3% BSA (Arnell, Fluka, etc.)� 15 g

0.02% NaAzide� 5 ml 2% Azide (LEAVE OUT FOR ECL)