Hello!
I've been producing some monoclonal antibodies with an unknown target.
As a quality control, I've been running a reducing SDS-PAGE on 12% gel (to check for contaminants and control the length of the chains) and a nonreducing SDS_PAGE on 7.5% gel (to ensure the chains form S-S bonds).
I got the opportunity to run analytical size-exclusion chromatography as well. Seeing as this allows me to check the antibodies in their most native form, I considered this the best method for QC.
But now I have some antibodies that do not look good on the gels, but pass the SEC with no problem.
How can this be? Is the antibody actually misfolded, or does it just react with SDS in a peculiar way?
Especially number 3 confuses me. It looks like the heavy chain aggregates after denaturation on the reducing SDS-PAGE. The non-reducing gel also does not look good.
The lane with a green tick is a control antibody that I know is correct.
In the attached SEC curve image (mAU normalized against the max mAU value of each curve), this same control antibody is represented as the red curve, and number 3 is the blue curve.
Indeed your sample 3 seems odd on SDS gels. In non-reducing gels, it migrates in different forms but it is not totally aggregated. In reducing conditions, it simply does not enter the resolution gel (a big band at the top can be clearly seen). Did you run both gels and made the SEC essentially at the same time (i.e. same sample's "age")? It could be that, due to e.g., unfolding, intermolecular S-S bonds could, as time goes by, lead to precipitation.
Hello Italo Augusto Cavini !
I agree, the aggregation that seems to happen only after the denaturation of the S-S bonds confuses me too. Although, the reducing gel is 12%, so it could be that the reduction to 7.5% let the aggregate enter the nonreducing gel.
The SDS-PAGE gels were run simultaneously (I have also done them twice, and have done a second transfection of this antibody - it looks the exact same).
The SEC was also run after the gels, so if it was due to precipitation over time I would have assumed to see it there as well.
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