![]() 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. Confirm the presence of protein by another method. The use of an antibody that reacts specifically with an epitope commonly introduced into a recombinant protein eliminates the need for a protein-specific antibody, and allows the use of one antibody for the detection of all proteins containing this feature. ![]() Increase the amount of total protein loaded on gel. Increase antibody concentration (2-4 fold higher than recommended starting concentration). Incubate with secondary antibody conjugated with HRP for 30 min at room temperature. Antibody may have low affinity to protein of interest.Wash three times with TBS-T (3 x 5 min).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.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. 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. ![]()
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