Hi All,
I am seeing protocols suggest including EDTA with a reducing agent when reducing disulfide bonds responsible for antibody accumulation and was just wondering how the EDTA's chelating action keeps thiol groups free and why this is necessary if an excess of reducing agent is still present to reduce possible future disulfide bridges.
Thank you!
EF
Hi Eduard,
EDTA as a chelating agent helps to avoid metal-induced oxidation of thiol groups.
Regards, Christian
That is what I came across but is that really a common occurrence? I figured there were so few metal atoms present that this could not be an issue...
Thanks,
EF
To my understanding the majority of these metals and their ions (including Ca2+ and Mg2+ which is what EDTA chelates) in such solutions stem from the buffers in which the antibodies are eluted during the purification (and conjugation) steps. For one, you need the right buffer conditions for elution of the proteins. Second, it's cheaper to just add EDTA than to try and 'purify' the eluate by making it metal-free.
Also, even though the concentration of these metals might be very low in solution, they're not being used up but remain available for continous oxidation (as Christian pointed out) and, hence, over time this would negatively affect antibody integrity/quality.
Cheers!
This is something I never considered too significant but now I know better, thank you gentlemen!
All the Best,
EF
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