This protocol can be used to prepare “blocked” antibody for use in either western blotting or immunohistochemistry.
Determine the optimal concentration of antibody for your protocol. Next, calculate the amount of antibody needed for two experiments (i.e. if the recommended western blot dilution is 1:1000 and 2 mL of antibody solution is required for staining, then 2 uL of antibody is diluted in 1998 uL of buffer.
Dilute enough antibody in blocking buffer to ensure enough reagent for two experiments (4 mL). Divide the diluted antibody into two tubes.
Label the first tube “Plus Peptide” and add blocking peptide at a ratio of 5:1 to 10:1 (i.e. if 1 ug of antibody was added in Step 1, then add 5-10 ug of blocking peptide).
In a second tube labeled “No Peptide” add an equivalent volume of saline/PBS, so both the peptide containing and non-containing tubes contain the same volume.
Mix gently and incubate both tubes at room temperature for 1hr or overnight at 4°C.
Proceed with the normal staining protocol on identical samples staining one set of samples with the blocked antibody and the other with the control antibody.
Compare staining patterns between the blocked and unblocked antibodies.
