Looking to label a enzyme (at it's -COOH groups) with an dye with an amine or cadaverine group. (I can't use an NHS ester here, two important sites of the enzyme would be compromised). There are lots of dyes available, but I'm surprised that I can't find a manual/protocol out there.
Basic gist is to mix protein with a large molar excess of dye (100:1) then start reaction by spiking EDC, stopping with Zeba column purification. Protein cross-linking is obviously a concern, hoping the large molar excess and a shorter reaction time help mitigate this problem.
Any advice/experience on buffer pH, ionic strength, EDC concentration, etc. for this reaction?
Answering my question here, in case someone stumbles on the same literature gap:
Called Molecular Probes. They advised to follow the normal EDC conjugation protocol (see manual for product PG82079). Based on the BSA conc., they're running at a 65ish time excess (dye to protein). Just be wary of this if you're going this route.
Hi Matt. I was wondering if you could reach a final protocol for this question. Which EDC concentration did you use? Which protein concentration? Thanks!
- Badges
- Science topic