I am trying to conjugate oligo modified in the center with amine to NHS-ester modified dye.
As oligo is modified in the center of the chain, the degree of labeling achieved is less than 40 percent.
I have been trying different buffers and different pH. But noting resulted in increasing the degree of labeling.
Please help me if there is something I am missing here.
Thanks in advance
pH is the key, should be between 7.5-8.5, I believe ~8.3 is the best. Is your 40% based on Oligo or based on NHS ester, I guess it based on Oligo.? 4 to 5 times of NHS ester is used for labeling according to standard protocol (attached). You can always add more NHS ester to push the yield higher, though sometime it may not work that well. We got higher than 60% routinely.
Good luck!
If you want to produce an amide bond from NHS Ester and and amine
By using Tri ethyl amine as catalyasis
Ahmed Kareem Hussein
Use TEA or DiPEA, or else, in anhydrous organic solution only, not for in liquid phase.
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