Peptide Blocking Assay:
A peptide-blocking assay is useful for confirming specificity of a peptide antibody against the target protein. In many cases, an inhibition study such as this is a requirement in publications to confirm that the antibody is specific to the native protein.
There is some trial and error involved with determining the correct amount of peptide to use, but the general idea is to incubate affinity purified antibody with the peptide prior to running an assay such as a western to see if this eliminates bands against the target protein. If enough peptide is used, then all antigen-specific bands should be eliminated.
Here is the protocol that we recommend:
- Mix 5ul of peptide (at approximately a 1 mg/ml concentration) with 5ul of affinity purified antibody. The peptide can be dissolved in PBS.
- Let the peptide / antibody mixture sit for approximately 2 hours at room temperature.
- Dilute the antibody / peptide mixture to a working concentration of 1:500 for running on the western blot.
- Run 2 lanes of the same sample on the western.
- Assay one of the lanes against the purified antibody that has been diluted to a working concentration (e.g. 1:500)
- Assay the other lane against the antibody / peptide mixture that was prepared in the previous steps.
- Ideally, you will see the band on the lane that has been assayed with the purified antibody and you will not see a band on the lane that has been assayed using the antibody / peptide mixture (since the peptide is blocking the peptide-specific antibody).
- In some cases, there may simply be a reduction of the band and so it may be necessary to increase the quantity of peptide used in the first step in order to completely block the band. Some trial and error such as this is typical.
Next: Peptide Reconstitution