![]() ![]() Compare the signal from your unknown sample to that of the standard and estimate the concentration.Try several different lengths of exposure. Incubate with ECL reagent for 1 min, cover with Saran wrap (remove excessive solution from the surface), and expose X-ray film in the dark room.Wash three times with TBS-T (1 x 15 min and 2 x 5 min), then once with TBS (5 min).For optimum antibody dilution, follow the manufacturer's recommendation. Incubate with secondary antibody conjugated with HRP for 30 min at room temperature.Determine how much protein to load and add an equal volume 2X Laemmli sample buffer. Determine the protein concentration for each cell lysate. ![]() Wash three times with TBS-T (3 x 5 min). Remove a small volume of lysate to perform a protein quantification assay.Incubate with primary antibody (0.1–10 µg/mL for purified antibody, 1:1,000–1:100,000 for antisera, 1:100–1:10,000 for hybridoma supernatant) in BSA/TBS-T for 30 min at room temperature.Sample is added (0. Capture antibodies are supplied arrayed/spotted on a membrane with each pair of spots representing a different analyte. Block non-specific sites by soaking in 5% BSA in TBS-T in a 10 cm Petri dish (30 min to 1 h at room temperature). Antibody arrays are an antibody-pair-based assay, analogous to ELISA, but using a membrane as a substrate rather than a plate.Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly. Using a narrow-mouth pipette tip, spot 2 µL of sample onto the nitrocellulose membrane at the center of the grid.Draw a grid by pencil to indicate the region you are going to blot. Have the nitrocellulose membrane ready.Dot blot is a technique for detecting, analyzing, and identifying proteins, similar to the western blot technique, but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate. Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc.) Dot blot protocol General dot blot procedure. A technique for detecting, analyzing and identifying proteins, similar to the western blot technique but differing in that protein samples are not separated electrophoretically but are spotted through circular templates directly onto the membrane or paper substrate.Ĭoncentration of proteins in crude preparations (such as culture supernatant) can be estimated semiquantitatively by using the dot blot method if you have both purified protein and specific antibody against it. ![]()
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