PKA-alpha-Western-blot

1. SDS-PAGE

Make sure to load 5 ul of color protein marker

2. Transfer

use transfer buffer with 10% methanol

25 v for 1 hour

3. Blocking

TBST with some milk powder for 15 min, then rinse with water and TBST

4. 1st antibody

Rabbit anti-PKA, 1:2000, add 1 ul to 2 ml TBST, incubate (shaking gently) at RT for 1 h.

5. Wash

rinse once with water, then 10 ml TBST, 3 times, 5 min each

6. 2nd antibody

AP conjugated Goat anti-Rb, 1:10000, (0.5 ul to 5 ml TBST)

RT for 30 min gentle shaking.

7. Wash

rinse once with water, then 3 times of 10~15 ml TBST, 5 min each.

8. Develop

Rinse with water, add 5 ml of AP buffer, 50 ul BCIP/NBT Color Development Substrate.

stop reaction until you see brown bands.

Alkaline Phosphatase Buffer (AP Buffer)

100mM Tris-HCl [pH 9.0], 150mM NaCl, 1mM MgCl2)

NBT/BCIP

18.75 mg/ml NBT (Nitro blue tetrazolium chloride) and 9.4 mg/ml BCIP (5-Bromo-4-chloro-3-indolyl phosphate toluidine salt) in 67% DMSO