An Introduction to Blocking Assays: Pre-Absorbing Antibodies

One of the questions we get asked more than any other, is if the immunizing peptides used for a given antibody is available for purchase to be used in a competition assay or blocking assay. As useful as these are, few companies make them available for purchase. Luckily, Everest is one of the companies that do make their immunizing peptides available for purchase. Abcore now stocks the majority of these synthesized peptides in our San Diego facilities for immediately shipment to end users throughout North America.

Synthesizing peptides can be used in a blocking assay as follows: Everest antibodies ship at a concentration of 0.5 mg/mL, and the blocking peptides ship as a 0.1 mg lyophilized pellet. Reconstituting the peptide pellet in 0.2 ml of water makes the peptide concentration 0.5 mg/mL, the same as the antibody. With the concentrations now matching, mix identical volumes of the peptide and antibody together at ambient temperature for 20 minutes on a rotator. There will be a molar excess of peptide relative to antibody, so blocking of the antibody should be complete. Perform two western blots in parallel: one using primary antibody that has not been pre-absorbed with the immunizing peptide, and the second using the pre-absorbed primary antibody pre-absorbed as described. The specific band(s) which correspond to the target protein should disappear in the pre-absorbed western blot which serves as a confirmation of specificity relative to any background or nonspecific signal. Shown to the right are the results of 2 such pre-absorption assays.

Figure 1 is taken from catalog number EB06142, anti-ARHGAP26 antibody, and shows a western blot done without the blocking peptide (lane A) and with the antibody pre-absorbed with the blocking peptide (lane B). As you can see, both bands seem specific to the immunizing peptide and represent specific signal within the human heart lysate.

Contrasting with Figure 1, is Figure 2. Figure 2 is taken from catalog number EB05974, anti-USP6 antibody, also shows a western blot done without the immunizing peptide (lane A) and with the antibody pre-absorbed with the immunizing peptide (lane B). From this figure you can see that the ~90 kD band is competed off, confirming its specificity for the immunizing peptide. However, the ~50kD band remains meaning this is most likely a non-specific cross-reaction or is the result of the secondary antibody used.