You could use HABA dye (4'-hydroxyazobenzene-2-carboxylic acid) which is a reagent that enables quick estimation of the mole-to-mole ratio of biotin to protein in a solution. There is a kit available from Pierce/ thermo scientific for the same. Or you could just spot your biotinylated antibody antibody on a nitorocellulose membrane and visualize using Streptavidin-enzyme conjugate and colorimetric substrate. Another option is to do the same in ELISA well.

You could use HABA dye (4'-hydroxyazobenzene-2-carboxylic acid) which is a reagent that enables quick estimation of the mole-to-mole ratio of biotin to protein in a solution. There is a kit available from Pierce/ thermo scientific for the same. Or you could just spot your biotinylated antibody antibody on a nitorocellulose membrane and visualize using Streptavidin-enzyme conjugate and colorimetric substrate. Another option is to do the same in ELISA well.

Thank you Neeraja

Yes, HABA assay is very easy way to test this . Add approximately 50/500 mole excess of HABA to your streptavidin and incubate for 30 minutes at room temperature, this will give an absorbance at around 500nm in UV-Vis resulting from HABA bound to SA. If you add your biotinylated antibody to this solution in stoichiometric amount, this will cause decrease in absorbance at 500nm reflecting successful binding of biotin to streptavidin replacing HABA. ( Also it might be that your Biotin labeling was a success but western blot transfer was not)

Thank you Anindya. The problem is that I have to know whether

my biotinylation worked in the next few days. We currently do not have HABA in our lab and it would take at least 3 weeks for it to be shipped. Is there another way?