How to prove that thiol groups are de-protected?

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sergii
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How to prove that thiol groups are de-protected?

Post by sergii » Wed May 02, 2012 7:32 pm

Hi everyone.

I'm trying to link chemically a protein with a DNA oligonucleotide. I think one of the simplest way would be to use Michael addition, where thiol group reacts with C=C electron deficient bond. Befor trying to work with proteins, which could be hard, I'm trying to ligate two DNA oligonucleotides, one modified with thiol group, another is modified with acrydite group:

DNA1--SH + CH2=CH--C(O)--DNA2 ---> DNA1--S--CH(CH3)--C(O)--DNA2

Thiol-modified DNA comes in protected form, like this: DNA1--S--S--ProtectionGroup. Before performing the reaction of interest, one should deprotect thiol group, which is done by reaction with DTT and further desalting.

The reaction I'm trying to do fails, and I don't know whether the reason for faiure is Michael addition itself, or failed deprotection, or failed desalting.

So here my question comes: is there any simple way to test, that deprotection was succesfully done and DTT was completely removed from the system?

Thank you very much for help!

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jonmoulton
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Re: How to prove that thiol groups are de-protected?

Post by jonmoulton » Wed May 02, 2012 10:10 pm

MALDI-TOF mass spec comes to mind to check if the disulfide is broken. I suppose it depends what instruments are at hand.

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JackBean
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Post by JackBean » Thu May 03, 2012 6:47 am

that was exactly my answer :)
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

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