ASR
(1) Run gels
One to stain and one to Western blot. Include the Rainbow makers
(2) Transfer
Fix gel in the transfer buffer 1 X 15' and the nitrocellulose membrane 1 x 15'
Set up the Hoeffer transfer tank: run 50V (~0.5 A, 4-5hrs) or 25 V (~0.2 A) for overnight
(+) Red grid - Scotch Pad - 2 x Filter- 2 x Membrane - Gel- 2 x Filter - Scotch Pad - Black grid (-)
Ensure all the bubbles are out
Starting Amps__________ Ending amps________
(3) Block
Place membrane in tray. Add ~100 mL TBS/Tween with 5% PDM 1 hr at room temp or 4°C o/n
(4) Primary Antibody
70,92,105,&124 (anti-D)
33,51,175,155 , 219 &236 (anti-N)
Incubate ~1hr with primary antibody 1-50 ug/ml total antibody (1µl each) in TBS
Use 30-50 mls
(5) Wash membrane
4 x 5' with TBS + 0.05% Tween 20 (~50 mls)
(6) Secondary Antibody
Incubate ~30' with the secondary antibody (goat anti-mouse horseradish peroxidase) 1:10,000 dilution in TBS + Tween
(7) Wash membrane
4 x 5' with TBS + 0.05% Tween 20
(8) Chemiluminescence
Combine 2 ml of Solution 1 and 2 and spread over membrane
Drain off after 1 min.
Place membrane between two overheads; exclude all air bubbles
Expose to film.
(9) Buffers
Transfer buffer: (5 L) or (1 L)
25 mM Tris (15.15 g) (3.03 g)
192 mM Glycine (72 g) (14.4 g)
20% MeOH (1000 ml) (200 ml)
Optional SDS inclusion:
**0.05% SDS 30ml of 10% SDS 3gm / 6 liters
** IF USING IMMPBILON-P USE 0.01% SDS !!! 0.6gm/6 liters
Tris Buffered Saline: (2 L)
20 mM Tris (4.84g)
137 mM NaCl (16g)
pH to 7.6 (~3 mL of 12 M HCl)
Other:
5% PDM (powdered dry milk) is 5g / 100ml of TBS
10% is 10g /100 ml TBS
0.05% Tween-20 (0.5 mL/L)