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I want to quantify a biotin-protein conjugate after chemical biotinylation. After biotinylation, the solution contains both biotinylated protein and free biotin.

I intend to use a HABA-avidin biotin quantitation kit like this one from Pierce. Since the biotinylated protein and the free biotin will both displace HABA and decrease the absorbance, I need to remove the free biotin so that I only quantify the biotinylated protein.

How do I separate the free biotin from the solution?

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I did some bioconjugation with NHS and maleimide biotins to proteins, peptides, and oligosaccharides. I removed the free biotin using spin filters or dialysis. Just be sure to use a MWCO well below the mass of your protein. I even used prep scale HPLC to remove biotin from the oligosaccharide. The HABA kit works well, I think I used that same one to quantify peptide - avidin linking. But try to keep your biotin protected from light and oxygen, it oxidizes fairly easily, I was getting +16 and +32 masses on LC-MS and couldn't explain it until I found some 50 year old paper on biotin sulfoxide. The oxidized biotin doesn't bind avidin nearly as well as normal biotin.

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You could precipitate the protein with alcohol (or acetone) (of course it will probably denature the protein), centrifuge the mixture and redissolve the protein to determine the amount of biotin bound to the protein.

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