Western Blot Video: SDS-PAGE Separation of Proteins Please contact Bio-Rad’s Technical Services Department to learn about recommended secondary reagents for specific applications. It may be useful to include a sample in which no primary antibody is used at all, in order to determine any nonspecific binding of the secondary reagent to the target tissue. 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.Īdd appropriate enzyme substrate solution and incubate as recommended by the manufacturer to visualize protein bands.Īppropriate controls should always be carried out. 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.
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