How do I remove free biotin after biotin-protein conjugation? 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?
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.