Sun Jul 19, 2015 8:58 am by hluche
Hi all,
Wonder if that means that the antibodies are getting over-reduced and split in half or that the light and heavy chains are separating
It probably does for some antibodies:
It happens that we ran some antibody before and after MAXPAR conjugation on a native and denaturing PAGE gel as a control for labeling by MAXPAR kit of another class of molecule.
We see at least four forms of polymer grafted onto the antibody after conjugation. On the native gel, we also see some light poly-peptide that could possibly be the light chain released from the full antibody (although we had no size marker for native gel) due to the reduction of S-S disulphydie bridge between H and L chain. On the gel, we also see a band of a slightly higher mass that could be due to polymer engraftment on some free L-chain.
The experiment is n = 1 and I don't intend to do this all the time to QC our antibodies as empirically labeling does work well for most of our antibodies. However, we did not pick this antibody for known poor protein recovery: it is an antibody that showed good labeling efficiency and that we used as a control to monitor labeling of another class of molecule. I have been told this effect is also seen for antibodies labeled to fluorochromes for conventionnal flow but there is certainly a chromatography step after labeling and before conditioning in tubes that ensure you just get the labeled antibody without free floating L-chain. It certainly explain why we need to add way more antibody in ug for mass cytometry than in conventional flow to reach labeling in saturating conditions: some mAb of the pool are just not fonctionnal anymore due to altered structure upon conjugation. One of the key point to limit this is to strickly follow the protocol and do not incubate more than 20-30 min for the reduction phase with TCEP.
For that exact reason, I would not try to change the size of the spin column from 50K to 30K even though you increase your yield of protein.
You will certainly retain in your conjugation prep some free floating L-chain fragment conjugated to polymer that will increase your yield in protein but will not help as not linked to your antibody anymore. However, this could increase background in your assay so I am not sure if it is worth it. For these particular mAbs, you should test it of course and let us know if reducing the size of spin column to recover more protein is increasing background staining.
Hervé