WESTERN PROCEDURE
Day before:
Make 1 L of transfer buffer and store at 4°
C
Procedure
- Equilibrate gel in transfer buffer for 15 minutes.
- During equilibration, cut nitrocellulose to the
size of the gel; wet in water and soak in transfer buffer.
- For each gel, cut 2 pieces of 3 MM paper to size
of gel and soak in transfer buffer.
- Wet pads with transfer buffer.
- Assemble transfer unit as outlined below:
(Red) pos. pole>
clear plate> pad> 3 MM> nitrocellulose> gel> 3 MM> pad>
black plate> neg. pole (Black)
- Close the sandwich board and dunk into partially
filled transfer chamber.
- Put in "BioIce" and fill chamber to
top, but do not over fill.
- Run transfer at 100 volts for 1 hour and no more.
This will require hundreds of mAmps and a special power supply.
- Verify transfer by staining with Ponceau S dye.
- Block with Blotto for at least 20 minutes at
RT°.
- Add appropriate amounts of primary antibody (e.g.
for purified mAbs; 5 µg/ml buffer).
For the antiserum: the antibody should be diluted 1,000 fold
- Incubate for at least 1 hour at RT°, or overnight at 4°
C.
- Rinse blot with Tris-Saline 3 X 2 minutes.
- Add Fresh Blotto to blot and the appropriate
amount of correct secondary antibody (e.g. 1:2500 dilution of goat anti-mouse
antibody conjugated with HRP). Incubate for 1 hour at RT° .
- Wash blot 3 X 5 minutes with Tris- Saline at
RT°.
- Add HRP substrate (called True Blue) and develop
color.
- Stop development when signal to noise ratio is
satisfactory by putting the blots in distilled water.
Equipment:
Normal gel apparatus
Western transfer chamber and gel holder,
200 Volt power supply
Nitrocellulose
Western Blot Solutions
10X transfer buffer stock - store at 4° C
- 30.3 g Tris base
- 144.1 g glycine
- water to 1 liter
1X transfer buffer - store at 4° C
- 100 ml 10X stock
- 500 ml H2O
- 200 ml methanol
- water to 1 L
- chill to 4° C
Antibody blocking and hybe solution - store at 4° C
- 1X Tris-Saline
- 3% w/v BSA (bovine serum albumin; special directions for dissolving)
- 0.1% v/v Tween 20
10X Tris-Saline - store at 4° C
- 6.05 g Tris base
- 43.9 g NaCl
- water to 500 ml
Not for use with His Tag - Blotto (make fresh each
time) - store at 4° C
amount of reagent
|
final
concentration
|
7 g Dry milk |
7% Dry milk |
10 ml 10X stock |
1X Tris-Saline |
100 µl Tween 20 |
0.1% Tween 20 |
100 µl antifoam A |
0.1% antifoam A |
water to 100 ml for the final volume |
|
Ponsceau S - store at RT°
- 0.1% w/v Ponsceau S
- in 1% v/v acetic acid
Lab Schedule Outlined
Lab Schedule In
Context of Research Project
Molecular Biology Main Page
Course Materials
Biology Main Page
© Copyright 2002 Department of Biology, Davidson College,
Davidson, NC 28036
Send comments, questions, and suggestions to: macampbell@davidson.edu