SILAC in E. coli

Hi all,

 

I would like to perform a SILAC experiment in E.coli. does anybody have a good  method for it?

thank you in advance,

Carlo

Posted on 06-Mar-2014 11:13 CET

Hi Carlo,

I have asked a colleague from the MS group and post his answer below. I hope there  is something useful in the links that he gave me. I will forward the email to your address, so you cvan get the attached pdf too.

"I do not have a protocol for SILAC in E. coli specifically as I never used SILAC in E. coli myself but have some experience labelling E. coli with artificial amino acids.

Depending on the application, proteomics or recombinant protein production, I guess the label could differ according to the question. Although for quantitative proteomics Lys and Arg as a combination with Trypsin for digestion are most commonly used.

I attached a pdf from a protocol paper. And also the links below  to some papers in which E. coli is used can be of interest.

http://www.ncbi.nlm.nih.gov/pubmed/17920290
http://www.ncbi.nlm.nih.gov/pubmed/23590516
http://www.ncbi.nlm.nih.gov/pubmed/24224529

For the rest: Any minimal medium should work. The question is whether or not to use an auxotrophic or a prototroph and supress endogenous synthesis by adding the amino acids to the medium.

http://www.ncbi.nlm.nih.gov/pubmed/23592334
http://www.ncbi.nlm.nih.gov/pubmed/24223875

For proteomics the conversion of arginine to proline is a potential pitfall with several solutions, see links below:

http://www.ncbi.nlm.nih.gov/pubmed/18487603
http://www.ncbi.nlm.nih.gov/pubmed/17762871

Hope this enough info to start with,

Please tell your colleague to feel free to contact me if he has follow up questions."

Posted on 13-Mar-2014 12:09 CET
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