I am trying to find a working protocol to label some proteins (commercially purchased) with carboxylate modified fluospheres (20 or 40 nm size). I figured out that this can be done using a EDC sulfo-NHS assisted conjugation. Though I could find some references, I have few concerns.
(1) My protein concentration (0.4 mg/ml) is low when compared to the references.
(2) Protein is stored in phosphate buffer but contains glycerol (50%), DTT (10 mM) and Triton-X (0.15 %) and I am not sure whether these have any influence on the reaction.
(3) The size of my fluospheres can't be larger than 40 nm.
(4) What kind of purification method will be suitable after the conjugation process?
Any suggestions?
1. I believe the concentration is not a matter, just be ensure that the conc of NHS is higher than your protein.
2. The biggest problem is your buffer, it is almost impossible to do the coupling reaction within DTT and others. Because your NHS may coupled on thiols or glycerol. You have to either do a acetone precipitation (if denature protein is acceptable) or you can do dialysis or membrane filtering using amicon .
4.Again, after coupling, use amicon or dialysis for native state protein and acetone precipitation for denatured state.
Hey Clarence!
Can you elaborate why DTT and Glycerol interfere with EDC/NHS coupling?
I tried finding some reference but couldnt produce anything hinting that DTT or Glycerol react with the activated ester.
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