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.