METHOD for Western Blots

1. While your SDS-PAGE gel is running, make your transfer buffer and chill to 4°C. Check that you have a frozen buffer dam is ready (stored in freezer next to Shikha, top shelf to the right side--looks like rectangular white plastic cup --should be full of ice).

2. Precut 3MM filter paper to size (10 cm X 7.5 cm)--Need 2. Cut nitrocellulose (handle with gloves!) to 8.5 cm X 6 cm.

3. When gel is finished, recover it from the apparatus and cut off wells. You will need a glass dish in which to assemble the blot under transfer buffer (to avoid trapping bubbles). Assemble on GREY side of sandwich:GREY side of holderSoaked rough white sponge (brillo pad)3 MM paper, precut and prewetgelnitrocellulose (pre-wetted in transfer buffer)3 MM paper, pre-wetSoaked rough white sponge (brillo pad)WHITE face of holder

Make sure there are no air bubbles between filters and gel. Close the sandwich and clamp shut with the white clamp on top.

1. Place the gel/nitrocellulose sandwhich in the transfer apparatus with transfer module--prefill to about 2/3 with of cold transfer buffer (For the biorad system, you use the same tank that you ran your gels in, but with a red and black transfer module that lives in the drawer (gel room, third drawer under the hybridization oven). You will need a medium size (longish but not too fat) stir bar that will still spin when the module is inserted (IMPORTANT) and also place the frozen buffer dam in next to the module. Add enough buffer to fill but not overflow.

2. Transfer at 41 volts (about 160-180 mA) for 2 hours with stirring (cold room).

3. Retrieve the membrane. You may write on it to mark its polarity (pen is fine). The prestained molecular weight markers should all have tranferred except the largest one.

4. You may stain the membrane in Ponceau-S (useful if you need to see lanes in order to cut it) or directly place it in blocking solution: 1hr at room temp (gentle shaking) or overnight in fridge.

5. Wash off blocking solution with PBS-T, use 25-50 ml for 5 minutes, shaking, at room temperature. Repeat.

6. During the last wash, dilute your primary antibody in BSA/PBST (15 ml is enough for one blot in a little clear --square--box, use 25 ml for 2 blots). Pour off wash, then immediately add the primary antibody. Incubate at room temp, 60 minutes or overnight at 4C. Pour off.

7. Wash 4 X 10 minutes in PBS-T (25 ml to 50 ml per wash), shaking.

8. During last wash, dilute secondary antibody (goat anti-rabbit HRP at 1:2500 i.e. 10 ul antibody in 25 ml BSA/PBS-T). Pour off last wash and add antibody. Incubate 60 minutes, room temp with shaking.

9. Wash as before.

10. For ECL: Mix 5 ml enhanced luminol with 5 ml oxidizing reagent (these are stored in the dark at 4C...and must be returned to the cold promptly). Add this mix to the drained membrane and incubate 1 minute.

11. Blot excess liquid on paper towel and place membrane in a plastic sheet holder. Promptly take it to the darkroom and expose it to film. Try 10" seconds and then decide if you need longer or shorter.      Western blots, Biorad apparatus:      Transfer buffer (fresh): 100 ml 10 X running buffer      200 ml methanol      700 ml dH20      10X PBS: 80 g NaCl      2 g KCl      11.4 g Na2HPO4      2.4 g KH2PO4      adjust pH to 7.3, add dH2O to 1 liter. Autoclave.      1X PBS-T: 100 ml 10X PBS      1 g Tween-20 (it's a liquid, weigh in little boat)      900 ml dH20      makes 1 Liter (this keeps on shelf indefinitely)      Blocking buffer: 2.5 g powder non-fat milk in 50 ml 1X PBS-T (make fresh every time)      Wash buffer: 1X PBS-T (see above)      Antibody buffer: 3.33 ml 30 % BSA solution + 97 ml 1X PBS-T (make fresh every time)