I'm trying to make a conjugation carrier with KLH-MBS. My protocol is as:
1. 5mg of KLH+ 500ul of 10mM phosphate buffer (pH=7.2)
2. 1.5mg of MBS+ 100ul DMF (>99% purity)
3. 70ul of MBS from "2" is added to the KLH on "1"
4. Incubation the mixture on RT for 30min
But, every time when I add the MBS to the KLH, some particles form inside which indicates denaturation o the KLH protien. Can someone explain why?
My guess is that KLH is not soluble in the concentration of DMF you mixed into it.
Thanks for your answer Adam. I'm following this protocol from a Journal though nothing about DMF concentration is written there. Does anyone knows about DMF concentration please?!
The protocol is a regular one. Klh precipitation will always occur given its nature and solubility. Furthermore upon activation you reduce hydrophilicity of klh's surface. Continue with your conjugation and discard aggregates.
I have to take OD value with spectrometer (with DTNB buffer) which must be more than 0.3. but every time my OD value of the carrier is 0.015 or something. I think this is because of the KLH aggregation, maybe it gets denatured. I can't use this aggregated carrier for my peptide!
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