I'm just curious if blocking peptides can be used in this fashion? Does the conjugation of the blocking peptide with the primary antibody stop the immunoreactivity with say a flouro-tagged secondary antibody? Say I loaded a serial dilution of blocking peptide on a gel, ran it, incubated with primary then secondary, then imaged, should I expect to see a band?
If by "blocking peptide" you mean a peptide that corresponds to a continuous epitope recognized by the antibody, then if you can resolve it as a band on a gel and transfer it to the blot, you should be able to detect it with the antibody. The problem is that peptides are usually very small for SDS-PAGE. You may have to use a high percentage gel, a gradient gel, or a Tricine gel to prevent it from running off the gel.
The epitope-containing peptide should not interfere with binding of the 2o antibody to the 1o antibody. (However, if you conjugate the peptide to the 1o antibody, i.e. attach it covalently, it could interfere if the peptide:antibody stoichiometry is high enough to block multiple binding sites for 2o antibody. I assume this is not what you meant.)
If by "blocking peptide" you mean a peptide that corresponds to a continuous epitope recognized by the antibody, then if you can resolve it as a band on a gel and transfer it to the blot, you should be able to detect it with the antibody. The problem is that peptides are usually very small for SDS-PAGE. You may have to use a high percentage gel, a gradient gel, or a Tricine gel to prevent it from running off the gel.
The epitope-containing peptide should not interfere with binding of the 2o antibody to the 1o antibody. (However, if you conjugate the peptide to the 1o antibody, i.e. attach it covalently, it could interfere if the peptide:antibody stoichiometry is high enough to block multiple binding sites for 2o antibody. I assume this is not what you meant.)
Ah. Thank you for the clarifications. Very helpful.
Kind regards,
Martin
Blocking peptide is useful if you want to confirm the multiple bands on blot are specific signals (eg. due to existence of splice isoforms) and are not background noise.
Instead of running the peptide in the gel, I pre-incubated the primary Ab with (A) and without (B) blocking peptide before applying to the blot. Bands that disappear in (A) represent specific signals. Santa Cruz provides blocking peptides for some of the primary antibodies.
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