Procedure

1. Equilibrate gel in transfer buffer for 15 minutes.
2. During equilibration, cut nitrocellulose to the size of the gel; wet in water and soak in transfer buffer.
3. For each gel, cut 2 pieces of 3 MM paper to size of gel and soak in transfer buffer.
4. Wet pads with transfer buffer.
5. Assemble transfer unit as outlined below:
   (Red) pos. pole> clear plate> pad> 3 MM> nitrocellulose> gel> 3 MM> pad> black plate> neg. pole (Black)
6. Close the sandwich board and dunk into partially filled transfer chamber.
7. Put in "BioIce" and fill chamber to top, but do not over fill.
8. Run transfer at 100 volts for 1 hour and no more. This will require hundreds of mAmps and a special power supply.
9. Verify transfer by staining with Ponceau S dye.
10. Block with Blotto for at least 20 minutes at RT°.
11. 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
12. Incubate for at least 1 hour at RT°, or overnight at 4° C.
13. Rinse blot with Tris-Saline 3 X 2 minutes.
14. 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° .
15. Wash blot 3 X 5 minutes with Tris- Saline at RT°.
16. Add HRP substrate (called True Blue) and develop color.
17. 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

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