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 spalashing buffer and rolling 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, transfer for at least 1.5- 2 hours at 100 V constant voltage (this is for a mini-Transblot apparatus; large apparatuses require higher voltage and/or longer transfer times). Change ice pack 1/2 way through transfer.
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. Common dilutions:
--anti-1M92-7 1:1000 of protein G purified antibody
5. Wash 4 times five minutes with wash buffer.
6. Reblock the membrane for 1 hour on room temperature rocker in block solution.
6a. Incubate membrane for 1 hour with 2.5 mg/ml Rabbit anti-mouse IgG (H+L). Wash membrane 3 times (15 minutes) with block buffer.
7. Pour off block solution and add 125I-protein A (2 uCi/10 ml) in block buffer for 1 hour on room temperature rocker.
8. Pour off and save solution (125I-protein A solutions can be re-used several times). Wash membrane 4+ times (the more the better) 15-20 minutes with wash buffer.
9. 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.
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