Western Blotting Protocol

Reagents

Blocking Buffer

Block Ace BUF029 dissolved in water, or 5% non-fat dried milk dissolved in PBS.

*Note: For cleaner western blots, Block Ace is recommended over 5% non-fat dried milk dissolved in PBS. ​For the detection of phosphorylated protein, use the recommended blocking solution as stated on the product datasheet. However, we advise using our protocol for detection of phosphorylated proteins by western blot.

 

Washing buffer

Blocking buffer + 0.1% Tween 20

Ponceau S

Acetic acid, 5 ml
Distilled water, 95 ml
Ponceau S (Sigma P3504), 0.1 g

*Note: Ponceau S is light sensitive.

PBS

Disodium potassium phosphate, 1.15g

Distilled water, 1 L

Potassium chloride, 0.2 g

Potassium dihydrogen phosphate, 0.2g

Sodium chloride, 8.0 g  

PBST

Disodium potassium phosphate, 1.15 g

Distilled water, 1 L

Potassium chloride, 0.2 g

Potassium dihydrogen phosphate, 0.2g

Sodium chloride, 8.0 g
Tween 20, 1.0 ml
 

Method

1. Following SDS-PAGE, transfer proteins onto blotting membrane according to the manufacturer’s instructions.
2. Check protein transfer by staining the blot with Ponceau S for 1 min, then completely destain the blot by washing with distilled water.
3. Place blot into blocking solution for 2 hr at RT, or overnight at 4°C.
4. Rinse the blot briefly with wash buffer and then add primary antibody diluted in the wash buffer (a concentration of 1-10 µg/ml is generally acceptable, but check datasheets for precise recommendations). Incubate for 2 hr at RT, or overnight at 4°C.
5. Wash the blot extensively in wash buffer (3 x 10 min) with gentle agitation.
6. Add appropriate enzyme-conjugated secondary antibody diluted in wash buffer and incubate for 1 hr at RT with gentle agitation.
7. Wash the membrane with gentle agitation as follows: 4x 5 min in wash buffer; 3x 5 min in PBST and 2x 5 min in PBS.
8. Add appropriate enzyme substrate solution and incubate as recommended by the manufacturer to visualize protein bands.