Sean Lauber:Western Blotting

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Protein transfer to nitrocellulose

1) Prepare transfer buffer:

 For 1 L transfer buffer (enough to fill the apparatus):		
   10X Tris	        100 ml
   10X Glycine	        100 ml
   100% Methanol	200 ml
   ddH2O	        600 ml

2) Prepare 6 blotting papers (8.0 x 10.0 cm) for each gel

3) Prepare 2 sponges per gel

4) Prepare nitrocellulose membranes (5.5 x 8.5 cm) per gel

5) Remove the gel from the glass plates and cut off the stacking gel from the resolving gel

6) Soak these things in transfer buffer (and keep the resolving gel and nitrocellulose separate)

7) Prepare the apparatus - Black-sponge-3 filter-gel-membrane-3 filter-sponge-white - load it black to black

8) Assemble the resolving gel into a gel holder cassette and remove any bubbles (use a 15 ml falcon tube to roll them out)

9) Ensure the resolving gel is on the anode side and the nitrocellulose membrane is on the cathod side of the cassette (black to black)

10) Insert the gel cassette into the blotting apparatus in the proper orientation

11) Add transfer buffer, stirring rod, ice block, etc.

12) Transfer at 4*C for 1 h at 400 mA

Antibody incubation

For one blot, 100 ml TBS-Tween should be enough

1) Block the nitrocellulose membrane with 10 ml of 5.0% milk (dissolved in 1X TBS, 0.15% tween) for 1 h at room temperature

2) Incubate the blot with primary antibody overnight at 4*C (use 5% milk as diluent if Ab is polyclonal, 5% BSA if monoclonal, in TBS+tween)

3) Wash with TBS+tween 3x for 8 min at room temperature with rocking

4) Incubate blots with secondary Ab at room temperature for 1 hour (use 5% milk as diluent)

5) Wash blots 2x as before with TBS+tween

6) Wash blots 2x with TBS (NO tween) for 8 min at RT

7) Decant the TBS (as much as possible)

8) Add 3 ml of ECL immunoblot reagent per blot (1:40) and incubate for 5 min

9) Decant the ECL and dry the membrane as much as you can without directly contacting the surface

10) Place blots between plastic sheets and put into a film cassette to expose film in dark room