Hi! I am trying to conjugate a dye in its NHS ester form to the N-terminus of poly-glutamic acid peptide ( which is already available in the form of a white solid). I tried 3 times and am tring to monitor the extent of the reaction via TLC. However, I haven't been quite successful yet, hence, any suggestions would be helpful.
pH is the key for the conjugation, you should dissolve you dye NHS in anhydrous DMSO or DMF and then dropped into peptide in buffer(pH 8-8.5) with very fast stirring. TLC might be not the best way to monitor the conjugation, esp. for poly-glutamic acid peptide, HPLC would be much better!
Hi Guobing! Thank you for your suggestion. I tried dropping the polypeptide in a 0.1 (M) sodium bicarbonate solution whose pH was around 8.3. Also, I dissolved the dye in DMSO. I haven’t tried HPLC yet and I haven’t ever done it too. Could you kindly guide me what to expect if I run an HPLC?@
Depends on what kind of dye you have，dye can be very hydrophobic，also can be very hydrophilic. The best way is to run the HPLC of the dye acid/dye NHS ester, then run the rx mixtures, co-injection(of rx mixture and dye acid) if necessary to find out the retention time. You have to adjust the gradient of A and B(acetonitrile) buffer according the hydrophilicity of the dye, dye conjugate!
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes