Author: Unkown. Date: 6/1/00

Western Blot Protocol for PEP

1. Prepare 7.5% Acrylamide protein gel in hood.

11.4 mL H2O

3.5 mL acrylamide (40%)

5 mL resolving buffer

100 ul 20% SDS

100 ul 10% APS

10 ul TEMED

(Enough for 2 gels)

Layer with H20 saturated butanol (flammable cabinet)

Wait 30 min

Wash butanol out with H20

Kimwipe to dry butanol/H20

2. Prepare stacker

3.15 mL H20

1.25 stacking buffer

25 ul 20% SDS

25 ul 10% APS

5 ul TEMED

Fill to top of inner plate, insert comb (acylamide will spill out-be careful!)

Let polymerize 20-30 min

Take out combs

3. Assemble gel apparatus. Insert gel plates into plastic mold that has metal prongs. Once both plates are inserted, put them into small plastic holder. Add running buffer to middle section of apparatus and fill holder. Rinse out wells with the surrounding running buffer to remove unpolymerized acrylamide in stacking gel. Before loading samples, add 2-ME and heat at 90-100 degrees for 10 minutes. Load samples and MW marker (10ul kaleidascope marker w/6ul 6x loading buffer).

4. Run gel for 1hr at 200 volts or until dye is almost off the gel.

5. Transfer protein from gel onto Immobilon-P membrane. Wet membrane in methanol for 15 sec. Soak in ddH20 for 2 min. Put in transfer buffer for 5 min. Start sandwich with black side of cassette, then scotch brite pad, blotting paper, gel, membrane, blotting paper, scotch brite pad

6. Close cassette and assemble transfer apparatus with ice tray. Pour in half of the western transfer buffer. Put in sandwith and then pour in rest of buffer and a stir bar. Stir rapidly and run for 1 hr at 55 volts (or overnight at 30V in cold room).

7. Make washing buffer, and add milk to 1/2 of it--this is the blocking buffer.

Incubate blot with blocking buffer rocking overnight.

8. Incubate in primary antibody anti-HA bio, at 1 ug/mL rocking for 1 hour.

9. Wash blod in blocking buffer 4 x 5 minutes

10. Incubate in secondary antibody HA streptavidin at 2ug/mL for 1 hr.

11. Wash in washing buffer (without milk) 4 X 5 minutes.

12. Use Pierce chemiluminescent kit for signal detection. Add 3mL of solutoin A to 3 mL of solution B (let solution equilibrate to room temp during last washes), soak membrane in solution 5 minutes. Air dry. Cover blot fully in Saran wrap. Use markers for alighnment of film. Expose blot. to film.

Transfer Buffer

25mM Tris base 3.0 g

192mM glycine 14.4 g

10% methanol 100 mL

ddH20 900 mL

Washing Buffer

10mM Tris, pH 8 1.21 g

0.15 M NaCl 8.76 g

0.05% Tween-20 1 mL (50% v/v)

Save 500 mLs. To this, add 10 g non-fat dry milk (2% final) this is the blocking buffer