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. Introduce students to protein extraction, quantitation, separation, and analysis using techniques such as polyacrylamide gel electrophoresis (PAGE), western. 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. You can cast gradient gels through a bottom-filling port with the Model 485 gradient former to ensure reproducibility. Acrylic blocks act as space fillers when fewer than 12 gels are cast. Incubate for 2 hr at RT, or overnight at 4☌. Use the Mini-PROTEAN 3 multi-casting chamber to simultaneously cast up to 12 gels of 0.75, 1.0, or 1.5 mm thickness.
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 Wash the membrane with gentle agitation as follows: 4x 5 min in wash buffer 3x 5 min in PBST. Add appropriate enzyme-conjugated secondary antibody diluted in wash buffer and incubate for 1 hr at RT with gentle agitation. Wash the blot extensively in wash buffer (3 x 10 min) with gentle agitation. However, we advise using our protocol for detection of phosphorylated proteins by western blot. Incubate for 2 hr at RT, or overnight at 4☌. *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. The Mini-PROTEAN Tetra Cell can be configured for use with 1–4 precast or handcast gels in different thicknesses.Blocking Buffer Block Ace BUF029 dissolved in water, or 5% non-fat dried milk dissolved in PBS. Use the Mini-PROTEAN Tetra cell to teach protein fingerprinting, sample purity analysis, and downstream western blotting techniques in independent projects or.
BIO RAD WESTERN BLOT GEL SERIES
Protein Expression and Purification Series.Comparative Proteomics Kit I: Protein Profiler Module.Use the Mini-PROTEAN Tetra cell to teach protein fingerprinting, sample purity analysis, and downstream western blotting techniques in independent projects or with the following educational kits: Fast, efficient western blotting transfers Visualize proteins without staining in 5 minutes with stain-free option High resolution Stain-free option Best for 2DE High resolution For low MW proteins Best for 2DE Average Run Times: 1520 min: 15 min: 20 min: 60 min: 60 min: Gel Percentages: View migration chart to determine the best gel. An indispensable piece of equipment in research laboratories, the durable and easy-to-assemble Mini PROTEAN Tetra cell is also well-suited for use in classroom laboratories. Bio-Rad’s Mini-PROTEAN Tetra cell is a mini-format, vertical polyacrylamide gel electrophoresis system that provides rapid, high-resolution separation of protein (or DNA) mixtures by SDS-PAGE or native PAGE.
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