Immunopeptide blocking assay
Licia Miller Product Manager
Sometimes, antibodies may bind non-specifically to proteins other than the antigen. This is usually more common with polyclonal antibodies, but can also occur with monoclonal antibodies.
To determine which band or staining is specific, an immunopeptide blocking experiment can be performed . Prior to performing the staining protocol, the antibody is neutralized (incubated with excess peptide corresponding to the epitope recognized by the antibody). Antibodies bound to the blocking peptide are no longer available to bind to protein epitopes present on the western blot or in cells.
The neutralizing antibody is then used along with the antibody alone and the results compared. By comparing the staining with the blocking antibody to the antibody alone, you can see which staining is specific; this staining would not appear in a western blot or immunostaining done with the neutralizing antibody.
Materials and reagents
- Blocking buffer (usually TBST plus 5% nonfat dry milk or 3% BSA (for western blotting) or PBS plus 1% BSA (for IHC)
- Antibody blocking (immune) peptides
- Two pipes
- Two identical samples (e.g., a western blot with two identical lanes, cut in half; two slides containing cells of interest; etc.)
Experimental procedures
1. Determine the optimal concentration.
1.1 Determine the optimal antibody concentration that consistently produces positive results in a specific protocol.
1.2 Using this concentration, determine the amount of antibody needed for two experiments.
For example, if you have successfully used an antibody at a concentration of 0.5 µg/mL in a western blot. To stain one western blot using 2 mL of antibody solution, you would need to use 1 µg of antibody in 2 mL of buffer.
2. Dilute an appropriate amount of antibody.
2.1 Dilute the necessary amount of antibody in blocking buffer to the final volume required for two experiments.
2.2 Divide it equally into two test tubes. In the first test tube labeled "Blocking", add five times more blocking peptide by weight than antibody (in this case, 5 µg total peptide in 2 mL buffer). In the second test tube labeled "Control", add an equal amount of buffer.
3. Incubate both tubes with agitation at room temperature for 30 minutes or at 4°C overnight.
4. Staining. Perform the staining protocol on two identical samples, one with the blocking antibody and the other as a control.
5. Observe the staining. Staining that disappears when using a blocking antibody is specific to that antibody.
Tip: If more than one band disappears in a western blot due to peptide/antigen competition, these bands contain antigenic determinants and may be fragments of the intact antigen or complexes containing the antigen.
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