My purpose is to achieve more thiol (-SH) groups in an antibody without loosing its antigen recognizing property. So, we would like to cleave the disulphide bond at the hinge region which is holding the two heavy chains. This will give us two halves of the antibody which can still recognize the antigens. also the two halves will have -SH groups now.
We treated the antibody with 2-Mercaptoethylamine (2-MEA) of different concentrations (even higher than that suggested by standard protocols). But we didn't find any bands corresponding to the halves generated (at 75KD). Nor did we find the presence of -SH groups through the Ellmans reagent.
Can anyone suggest what could be the reason and how the reaction with 2-MEA can be worked out better.
Also, what would be the more reliable and reproducible ways to generate -SH groups in antibody without loosing its functionality.
Thank you very much for your time.
To my knowledge, there is no general, reliable protocol for this purpose. It depends very much on the structure of the antibody, under which conditions the cleavage has to be performed and whether this kind of cleavage works at all. Many researchers prefer TCEP for the reduction of disulfide bridges since this reagent seems to be more reliable. The major question is, for which purpose do you want to cleave the antibody? There may be better and simpler ways, e.g. to label an antibody.
See attached link. You also might want to look at the citing articles.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2539111/pdf/nihms-63637.pdf
- Badges
- Science topic
- Similar topics
- Chemistry, Organic
- Organic