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)