![]() Place the membrane in primary antibody solution diluted in 1% BSA in Tween PBS, pH 7.2, and incubate at room temperature for 1 hour with shaking. Wash the membrane with washing buffer 3 times (5 minutes each time). Place the membrane in blocking buffer and incubate at room temperature for 1 hour (or at 4☌ overnight) with shaking. Of the antigen by HRP reaction) ※An example performed at MBL Probing with antibodies and detection of the bands (blocking and detection Ponceau S is commonly used for this purpose because the membrane is easily destained, and it does not interfere with subsequent probing with antibodies. Turn off the power supply, remove the membrane, and temporarily stain to check the efficiency of transfer and to visualize the molecular weight markers to confirm their positions. *Transfer conditions are different depending on experimental conditions. Turn on the power supply and transfer for 1 hour at 50 mA (for one gel)*. Place a piece of equilibrated filter paper on the anode plate, and place the membrane, gel, and another piece of filter paper without any air bubbles.Ĭonnect the plus electrode to the anode plate and connect the negative electrode to the cathode plate. Skip using methanol, and equilibrate the membrane in transfer buffer.Įquilibrate the gel in transfer buffer after electrophoresis. ※There is no need for pre-wetting a nitrocellulose membrane. Soak a piece of PVDF membrane slightly larger than the gel in methanol for 1 minute (pre-wetting), and then equilibrate in transfer buffer.Īlso equilibrate two pieces of filter paper slightly larger than the PVDF membrane in transfer buffer. This video demonstrates SDS-PAGE separation of proteins using the Bio-Rad Comparative Proteomics Kit II: Western Blot Module.Īssembly of the blotting sandwich and electroblotting are shown along with the steps for protein detection using a colorimetric assay.Transfer of proteins to a membrane (semi-dry type blotting and membrane staining) 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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