I have some problem with the biotinylation protocol for a protein and I need your help.
I bought from Pierce a "Sulfo-NHS-LC-biotinylation kit" and I used it to incorporate the biotin into my protein (fibronectin).The kit I bought was for the biotinylation of 1-10mg of protein as weel as the protocol.
However, I used it to target a protein ammount inferior to that reported into the protocol. Maybe for this reason I encountered some problem regarding the incorporation. After the quantification of mol of biotin per mol of protein, I determined a great ammount of biotin into the sample.
Something goes wrong in the purification step?How can I check the quantity of unreacted biotin in my final sample?
Do you have some experience with this kind of biotinylation kit?Please find attached the file of the biotinylation protocol.