![]() ![]() ![]() The primary antibody is used following the manufacturer’s advice. The membranes are incubated in appropriate dilutions of primary antibody in TBST (150 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5) for 1 hour at room temperature or overnight at 4☌, with carefully shaking.After transfer, the membranes are blocked with a BSA blocking solution (5% BSA, 150 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5) or a milk blocking solution (5% powdered milk, 150 mM NaCl, 0.05% Tween 20, 20 mM Tris-HCl, pH 7.5).All of the remaining steps are carried out at room temperature. The protein transfer is carried out at 100 V for 1 hour at 4☌ with buffer recirculation. The transfer is carried out in transfer buffer (192 mM glycine, 20% v/v methanol, 25 mM Tris-HCl, pH 8.3). Proteins fractionated by SDS-PAGE are electrotransferred onto polyvinylidene difluoride (PVDF) membranes.The Mini Trans-Blot Electrophoretic Transfer Cell system (Bio-Rad) is used for western blot experiments.Gels are either electrotransferred to PVDF membranes.Gels are electrophoresed between 100 and 150 V in running buffer (3% Tris-HCl, 14.4% glycine, 1% SDS) until the bromophenol blue dye reaches the bottom of the gel.Samples are loaded and fractionated on 8, 10, or 12% polyacrylamide gels along with a protein ladder.SDS-PAGE is performed using the Mini-PROTEAN III electrophoresis system (Bio-Rad). The standard curve is produced by using varying concentrations of bovine serum albumin (BSA). The reading obtained is compared to a standard curve that allowed for a relative measurement of protein concentration. The Bradford assay method (Bio-Rad Protein Assay) is utilized to determine the concentration of proteins in each sample. Usually, samples are resuspended or mixed in 20-50 µl of 1X sample buffer and all the mixtures are incubated at 90☌ for five minutes or 50☌ for 15 minutes to break down the interactions between peptides. The schematic diagram of western bolt protocol. ![]()
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