Troubling for antibody biotinylation - (Jul/19/2012 )

Hi Everyone,

I recently did antibody biotinylation with pierce NHS-SS-biotin reagent. On western blot, antibody seemed to be biotinylated. i tried to find a way to quantify the biotinylation level.

Just any one know roughly how much biotin will be on one antiboy/??? (mouse IgG monoclonal antibody.)

Thanks.
Hong

Hi. Hopefully you've already found an answer to your question but incase you haven't......you can do a HABA assay. Sigma (as well as others) sells a HABA/avidin reagent that allows you to use a spec to measure the displacement of HABA molecules by your biotinylated antibody by the change in absorbance. You can then caculate the number of available biotins per antibody molecule. It is a rough estimate but a helpful one and will give you confidence that your protein was properly biotinylated.

Good luck!