Hey Dhiraj, I saw this email yesterday, and I was waiting for others to respond. This seems like there is no simple answer to your question. This would be advantageous, if you kindly share the chromatograms of the WT proteins, and the mutant one, both in the presence and absence of the reducing agent. Likewise, an image of Native PAGE, and the experiment conditions will help anyone to understand the complexity of the situation. Of course, you may hide the names of the proteins.
Kind regards, -Z Zaigham M Khan, PhD Associate Scientist Icahn School of Medicine at Mount Sinai Department of Oncological Sciences 1470 Madison Avenue New York United States On Sun, Nov 1, 2020 at 11:53 AM Srivastava, Dhiraj < [email protected]> wrote: > Hi Jon > there is no surface exposed cysteine nearby which can > potentially make disulfide bond with the modified cysteine > intramolecularily. > > Dhiraj > ------------------------------ > *From:* Jon Cooper <[email protected]> > *Sent:* Sunday, November 1, 2020 7:41 AM > *To:* Srivastava, Dhiraj <[email protected]>; > [email protected] <[email protected]> > *Subject:* Re: [ccp4bb] Cysteine oxidation product > > Hello, > > does the protein you added cysteine to (I assume you mean by mutagenesis) > have another cysteine which could make an intra molecular disulphide with > it? That would probably not show on non-reducing SDS? > > Best wishes, Jon Cooper > > > > -------- Original Message -------- > On 31 Oct 2020, 15:58, Srivastava, Dhiraj < [email protected]> > wrote: > > > Hi All > I added an extra cysteine residue to one of my protein > interactions partners and somehow it increased the affinity in the absence > of reducing agent however in the presence of reducing agent, the affinity > is same. there is no disulfide bond formation between the two interaction > partner as in non-reducing SDS-PAGE, I see the molecular weights > corresponding to only the individual proteins and not the sum of two. > Does anyone has ever seen this? at pH 8.0, what is the probability that a > surface exposed cysteine will have a net negative charge and still not very > reactive? can it exist as thiolate, sulfinate or sulfonate ion? is there > any such example in literature that anyone knows? > > Thank you > Dhiraj > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
