Hi All,
I want to reduce coupled Anti-EGFR mAbs for use in active-targeted nano-constructs. I want to use TCEP*HCl as my reducer but know that it causes (and actually needs) an acidic pH when used even in 1x PBS.
Would the pH caused by a 1 milliMolar (or around 100x to the mAb) concentration of TCEP*HCl be enough to harm my antibody?
Has anyone had experience reducing mAbs with TCEP?
Help is greatly appreciated!
Thank you,
EF
I don't think that 1 mM TCEP-HCl will change the pH significantly in a buffered solution, as long as the buffer concentration is high enough. It might be a problem with 10 mM phosphate, but would probably be OK at 50 mM. TCEP isn't very stable at neutral pH in phosphate, apparently, so add it from a stock solution of a lower pH. Or use HEPES.
Hi Adam,
The thing about TCEP is that it seems to actually require an acidic environment to function (as seen in "Scheme 1", page 2, attached paper), so I think raising the buffer concentration to keep the acidity in check would cause the reduction not to work...
Thanks,
EF
http://pubs.acs.org/doi/pdf/10.1021/jo00008a014
According to this, TCEP can be used at pH up to 9 for disulfide reduction.
http://www.biosyn.com/tew/instruction-of-reduction-reaction-using-tcep.aspx
Hmm that is strange, the Harvard paper showed that its mechanism relied on free protons...I can definitely try it though: maybe even a small number would be enough.
Thank you!
EF
Even at pH = 9 there are "free" protons, just at a much lower concentration. Reaction may be much slower at hight pH?
Check out the ThermoFisher Pierce website
https://www.thermofisher.com/order/catalog/product/20490
I think it has all the answers you seek.
Myron, I have seen the data sheet, all it says is that molecule reduces in around 5 minutes at a pH of 5. It does not address reaction rates at higher pH values.
Thanks,
EF
https://tools.thermofisher.com/content/sfs/manuals/MAN0011306_TCEP_HCl_UG.pdf
This seems relevant also
http://static1.squarespace.com/static/55c65df3e4b0bc58bd0bb80a/t/55d54774e4b04a41570d620d/1440040820078/Biochemistry+2004+Cline.pdf
Hmm these papers definitely imply the reduction would be just fine in a wide pH range...
That is great, however, I am now confused as to why every vendor I see packages it as a hydochloride salt? Is this just for the sake of water solubility and one is expected to raise the pH after dissolving?
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