3 replies to this topic

[SF_HK]

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?

[bob1]

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

IgG is normally about 150 kDa.

[knuf]

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

[bob1]

Whoops, yeah, what kfunk said...