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.Wash three times with TBS-T (3 x 5 min).For a 1x solution, mix 1 part of the 10x solution with 9 parts distilled water and adjust pH to 7.6 again. Pour off the block buffer, but keep membrane. Block the membrane with 5 dry milk in TTBS (50 mM Tris, 0.5 M NaCl, 0.05 Tween-20, pH 7.4) for 1 hour at room temperature. Ensure that the blots are dry before going to the next step. Incubate the membrane for 1 hour at room temperature. 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. Add distilled water to a final volume of 1 L. Blot 10 µl of 100 µg/ml of primary antibody onto membrane.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).Minimize the area that the solution penetrates (usually 2–4 mm diameter) by applying it slowly. Dot Blot protocol A technique for detecting, analyzing, and identifying proteins, similar to the western blot technique but differing in that protein samples. 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.Pre-adsorbed secondary antibodies - minimize non-specific binding and high background staining. Browse our large catalogue and resources. Nitrocellulose membrane (BIO-RAD, Trans-Blot, etc.) High quality HRP conjugated secondary antibodies for western blot, immunohistochemistry and ELISA. 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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