Make 1 L of transfer buffer and store at 4°
- 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
- 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°
- 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
- Add HRP substrate (called True Blue) and develop
- Stop development when signal to noise ratio is
satisfactory by putting the blots in distilled water.
Normal gel apparatus
Western transfer chamber and gel holder,
200 Volt power supply
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
| 7 g Dry milk
|| 7% Dry milk
| 10 ml 10X stock
| 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
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© Copyright 2002 Department of Biology, Davidson College,
Davidson, NC 28036
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