pbst wash removes protein ladder western blot

Wash the blot extensively in wash buffer (3 x 10 min) with gentle agitation.Īdd appropriate enzyme-conjugated secondary antibody diluted in wash buffer and incubate for 1 hr at RT with gentle agitation. Incubate for 2 hr at RT, or overnight at 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). Place blot into blocking solution for 2 hr at RT, or overnight at 4☌. 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 gįollowing SDS-PAGE, transfer proteins onto blotting membrane according to the manufacturer’s instructions.Ĭheck protein transfer by staining the blot with Ponceau S for 1 min, then completely destain the blot by washing with distilled water. Washing buffer Blocking buffer + 0.1% Tween 20 Ponceau S Acetic acid, 5 ml

However, we advise using our protocol for detection of phosphorylated proteins by western blot.

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

Pbst wash removes protein ladder western blot