Western Blot Experiment: Hoeffer and ECL

CHP

(1) Run gels

Run one to silver stain and one to Western blot. If SDS gels, include the Gibco BRL rainbow mw markers

(2) Transfer

• Fix gel in the transfer buffer 1 X 15' and fix the membrane 1 x 15'
• (If using Immobilon-P membrane, first wet in MeOH then transfer to buffer 1X 15')
• Set up the Hoeffer transfer tank: add buffer, insert sandwiches, and run 100 mAmp 30 V for overnight
• Order for sandwiches:

(+) Red grid - Scotch Pad - Filter- Membrane - Gel- Filter - Scotch Pad - Black grid (-)

Ensure all the bubbles are excluded!

Starting Amps__________ Ending amps________

(3) Block

put the best membrane into TBS with 1% BSA and 5-10% PDM for 45-60 min

(4) Primary Antibody

70,92,105,&124 (anti-D)

33,51,175,155 , 219 &236 (anti-N)

Incubate 45-60 min with primary antibody (1-50 ug/ml total antibody) in TBS +1% BSA or 0.25% PDM

(5) Wash

4 x 5" with TBS + 0.05% Tween 20 (50 ul/100ml) with 0.25% PDM

(6) Secondary Antibody

Incubate 30min to 1hr with the secondary antibody (goat anti-mouse horseradish peroxidase 1:10,000 dilution in TBS + 1% BSA or 0.25% PDM)

(7) Wash

4 x 5" with TBS + 0.05% Tween 20 (50ul/100ml) with 0.25% PDM

(8) Chemiluminescence

(Follow directions for specific kit) For ECL kit:

1 ml of Solution 2 spread over membrane

1 ml of Solution 1 spread

Wait 1 min., drain off

Wrap membrane in saran wrap or put between two overheads. Make sure all air bubbles are excluded. Expose to film.

(9) Buffers

Transfer buffer:

To make 1.5 L:

20 mM Tris (4.5 g Tris)

144mM Glycine (21.6 g)

20% MeOH (300 ml)

Carol's Recipe for 6L Transfer buffer for Hoeffer tank:

20 mM Tris (14.5 g Tris Base)

150 mM Glycine (67.6 g)

20% MeOH (1200 ml)

**0.05% SDS (30ml of 10% SDS or 3g / 6 L)

** IF USING IMMPBILON-P MEMBRANE, USE 0.01% SDS !!! (0.6gm/6 liters)

Tris Buffered Saline (10X):

0.5M Tris pH 8 (30.25 g Tris base)

1.5M NaCl (43.5g NaCl)

Milli-Q to 500 ml