I would like to perform a crosslinking experiment using BS3 crosslinker. My protein has 3 cysteines, but no S-S bridge, so I have to keep my protein in buffer with 5mM DTT. Can I perform the crosslinking experiment with DTT in my buffer? Does it somehow have affects on the crosslinking reaction? I couldn't find any information about the harmful influence of DTT in BS3 crosslinking, but before buying a reagent for $1000 I suppose it is better to ask.
Dear Elliot - I have performed BS3 crosslinking experiment with phosphate buffer and 5mM DTT - everything went well. DTT does't interupt BS3 crosslinking indeed.
BS3 can be used in the pH range of 7 to 9. pKa of cystein is about 8, can you perhaps conduct the the cross-linking reaction at the pH where the cystein is protonated (or mostly close to) ? Perhaps you can exclude DTT by making the cystein less reactive.
I found this paper: http://www.pnas.org/content/96/5/1892.full.pdf . They used mercaptoethanol and I got the impression that reducing agent did not interfere with cross-linking in their experiment. Perhaps you can follow their approach
I did cross-linking with BS3 to check for trimerization of my protein. In an SDS PAGE under denaturing conditions, the cross-linked protein stood as the trimer. So, the covalent bond form is stable.
Good luck.
Dear Wangsa thank you for your post. I supose BS3 doesn't react with cysteins but with primary amino grups - N-terminus NH2- and lysine side group. But if someone used BS3 with beta-mercaptoethanol I suppose DTT shoudn't be harmful. Thank you for the reply.
sorry Dawid, why you've to keep your protein in 5mM DTT?
BS3 is the water-soluble analog of the DSS,
and both react with primary amines, not sulfo-groups.
I lost something?
I usually use BS3 but I never needed use it in presence of DTT. Taking account that BS3 react with primary amines, as said Giuseppe Ossolengo, I think you can use it without problem.
I suppose there is some kind of misunderstanding. My protein has 3 cysteine groups and presence of DTT is required, otherwise it starts precipitating. I want to check the interaction with other protein, which also requires DTT in buffer. I was worried about DTT presence during BS3 reaction with primary amines of my proteins. But now I see that there is nothing to worry about.
A non-thiol reducing agent you might be able to use in place of DTT if it interferes with the BS3 reaction is TCEP.
Hi Dawid. I was wondering if you found your answer, and how your experiment went. You could desalt / buffer exchange with spin columns prior to your crosslinking reaction. This is preferred because of its speed. I would guess that precipitation (due to disulfide formation) does not take place immediately at 4 C or even room temperature. Intermolecular disulfide crosslinking rate can also lowered by reducing the protein concentration some in your sample, if this is an issue. Furthermore, I would think that crosslinking with BS3 would have the same potential for protein sample precipitation. After your crosslinking reaction, in your quenching step (glycine, Tris, etc) you could also add DTT or TCEP to retain free cysteine thiols (-SH) in your sample.
Here is some additional information and a general protocol for crosslinking with Bissulfosuccinimidyl suberate (BS3): http://www.covachem.com/bs3-crosslinker-82436-77-9-bis-sulfosuccinimidyl-suberate.html
Dear Elliot - I have performed BS3 crosslinking experiment with phosphate buffer and 5mM DTT - everything went well. DTT does't interupt BS3 crosslinking indeed.
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