My student and I tray to obtains Fc and Fab fragments from whole human IgG subclass whit this experimental conditions: ratio papain / Ab (3% ) (w/w), reaction time (24h), final volume (150µL) and papain activation time (30min) (this are the best condition we found in several analysis) but the digestions are incomplete and appear two additional bands in electrophoresis in some samples. Then what parameters or conditions we would change to optimize the digestion of IgG with papain? We need obtain these fragments with high purity for subsequently deglycosilation.
Have you already tried a protocol and saw more fragmentation than expected?
yes, we see more fragments than the expected. one around 55 kDa and other around 35kDa. this in native conditions, without denaturing reagents. Thank you Grant.
This is not uncommon. There are many parameters to optimize and the cleavage may still not be adequate. The condition we found would vary with different antibodies and was not an efficient use of time in an industrial setting so a completely different approach was taken where engineering was involved. The methods are company confidential but you might have to continue with an empirical approach as well as getting feedback from others for your specific antibody. Have you tried using different vendors for your source of papain? Temperature may also be an important variable in your partial factorial design. Do you know the cleavage site sequence for the 55 and 35 kDa peptides? Is the antibody recombinant or purified from human serum and of only one subclass of IgG? I assume the glycosylation pattern is homogenous and you are not using solid phase cleavage? Have you looked at alternatives to papain?
We try too with pepsin in parallel, and the result are more clean in electrophoresis after check de digestions, but so far we has not been able to obtain a glycosylation pattern characteristic for the each fragments (Fab and Fc).The sample analyze in both cases is a pool of purify IgG, isolated from human serum, this included all class of IgG. We only try with papain from Sigma because it is the only one we have available in our lab. In this time we do the deglycosilation in gel, but I think the critical step for whole process is the digestion of protein. Thanks for your ideas Grant. I hope receive more comments about it, to try with my sample.
This may be of interest to you if the digestion is critical and glycosylation studies are the final goal. The enzyme is very pure and specific. You might have already heard of it or even used it? Are you using LC/MS with triple quad mass spec? Do you have a positive control for the in-gel deglycosylation?
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