Separated proteins are transferred from the gel to a membrane where they are immobilized. Good sample preparation techniques ensure proteins remain undamaged for downstream analysis.Įlectrophoresis separates the proteins in the sample and provides molecular weight data for detected proteins during subsequent detection. The membrane is immediately scanned using an imager following manufacturer’s instructions.Need help with western blotting? Contact a SpecialistĬells containing your protein of interest must be lysed completely to ensure a high yield while removing non-protein components of cells.The antibodies are visualized by mixing the chemiluminescence development substrates, Peroxide Buffer and Luminol/Enhancer Solution (Pierce), in a 1:1 ratio and incubating this mixture on the membrane for 5 minutes.For chemiluminescence detection, the secondary antibody, horseradish peroxidase-conjugated α-mouse IgG, is incubated with the membrane at a 1:10,000 dilution in TBST for 1 hour.The reactions are terminated by washing the membranes in water. The antibodies are visualized by adding the color development substrates, Nitro blue tetrazolium (NBT) and 5-bromo-4-chloro-3-indolylphosphate (BCIP), in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl2, 100 mM Tris-HCl, pH 9.5).An additional 30 second wash with TBS is carried out.The membrane is washed with TBST as before to remove any free secondary antibody.For colorimetric detection, the secondary antibody alkaline, phosphataseconjugated α-rabbit IgG, is incubated with the membrane at appropriate dilution in TBST for 1 hour at room temperature. ![]()
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