peptide blocking - (Sep/12/2011 )

HI,

I need to perform a peptide blocking experiment for my antibody for western blot. I have never performed this and would appreciate any feedback.

I've read that the blocking peptides are used 10-100 times in excess of the antibody molarity? My antibody concentration is 2.5mg/ml and I use 1:250 dilution in TBST 5% milk for western blot. However I do not know how to calculate the amount of peptide to use.

My peptide concentration is 1mg/ml and has a calculated MW of 1876.55 Da. How much do I add to the TBST 5% milk?

You can assume a 1:1 binding of the antibody to the peptide.

IgG is normally about 150 kDa.

Actually, I think you should assume a 2:1 binding of peptide to antibody because each antibody has two binding sites.

Whoops, yeah, what kfunk said...