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