I performed a reaction to modify BSA through esterification, but it didn't seem to work. Most of the papers that I've found that attach azide to BSA seem to use maleimide. Is there a reason why NHS esterifcation potentially woudn't work? Thanks in advance
It should work, assuming that BSA has at least one surface-exposed lysine side chain to react with NHS. It is essential that the reaction occur in a buffer that is free of competing amines (for example Tris). Dissolve the BSA in a fresh solution of 0.1 M sodium bicarbonate. If it is already dissolved in an unsuitable buffer, then dialyze it thoroughly to remove that buffer.
Yes, it should readily react. Aside from the buffer requirements noted above, please be aware that NHS esters are unstable if not kept dry and cold (-20). You say that the reaction hasn't worked? Why do you draw this conclusion? Have you directly shown that azide groups have not been added? You could do this for example using a DBCO-fluorescent dye conjugate.
Another thing to note is the rate of reaction. The DBCO-azide reaction is very slow compared with for example maleimide-thiol. It would be unusual I think to add azide group using a maleimide, as this would require thiolation of the BSA, but I'm sure it would work. You still have the slow reaction with DBCO to follow of course.
Most likely explanations for failure are loss of NHS groups on storage, competing low molecular amines in the reaction, and insufficient incubation time with DBCO (or similar) group.
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