CHREBP Antibody Blocking Peptide

A: Here is a protocol for a blocking peptide control for a western blot: Materials and Reagents Blocking buffer (usually TBST plus either 5% non-fat dry milk or 3% BSA for Western blot, or PBS plus 1% BSA for IHC) - Antibody - Blocking (immunizing) peptide - Two tubes - Two identical samples (e.g. a Western blot with two identical lanes, cut in half; two slides containing the cells of interest; etc) Method 1. Determine the optimal concentration of antibody that consistently gives a positive result in your particular protocol. Using that concentration, determine how much antibody you will need for two experiments. a. For example, an antibody is being used successfully in Western blot at 0.5 ug/ml. You will need 2 ml of antibody solution to stain one strip of a Western blot. Thus, you would use 1 ug of antibody in 2 ml buffer for each strip. 2. Dilute the necessary amount of antibody in blocking buffer to the final volume needed for the two experiments. Divide this equally into two tubes. 3. In the first tube, labeled locked, add the blocking peptide to a final concentration of 1 ug/ml (2 ug total peptide in this example). In the second tube, labeled Control, add an equivalent amount of buffer. 4. Incubate both tubes, with agitation, at room temperature for 30 minutes, or overnight at 4 degrees C. 5. Perform the staining protocol on the two identical samples, using the blocked antibody for one and the control for the other. Be careful not to mix up the strips using the blocked and control antibodies! 6. Observe the staining. The staining that disappears when using the blocked antibody is specific to the antibody. (See note i) Notes i. If more than one band disappears in Western blot by peptide/antigen competition, those bands contain the antigenic determinants and could be fragments of the full antigen or a complex containing the antigen.