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methods or any paper to know how to incorporate radioactive sulphur in proteins

methods or any paper to know how to incorporate radioactive sulphur in proteins - Proteomics Forum

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Old 01-26-2008, 10:30 AM
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Default methods or any paper to know how to incorporate radioactive sulphur in proteins



hello all,
how we can apply radioactive sulphur during growth to detect it in proteins weather it is incorporatred in specific protein or not?i am interested in specific type of protein family
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Old 01-26-2008, 02:16 PM
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Cool Re: methods or any paper to know how to incorporate radioactive sulphur in proteins

Hi!
I have been an expert at S-35 protein labeling during my PhD.

It depends what you want to do. Do you want to assess the synthesis rate or degradation rate of a protein?

To assess synthesis rates of proteins, you pulse (add a small amount of S-35 methionine) and then wait 2-30 minutes depending on the size of your protein.

Then you can lyse your cells after pulsing, immunoprecipitate and run them on a gel after the IP. This would give you synthesis.

If you are assessing degradation, you can pulse then wash cells after a set time and then chase to assess degradation. There are many research papers on this method, however its not well known.

If you need any help, ideas or just to let us know what you are doing just post back here, would love to hear from you.

Cheers
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Old 01-29-2008, 09:52 AM
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Default Re: methods or any paper to know how to incorporate radioactive sulphur in proteins

Hi
Thanks for responding and giving good hints.I am working on plant proteomics and i want to observe proteins which are synthesized during that stress and i want to labell them with S35 methionine.i am only interested in those proteins which are differentialy expressed during that stress rather than whole proteins.
but we have to apply S35 methionine for only short period is it enough to labell all proteins which are upregulated in that time because they will be already synthesized before applying radioactive material then how in short period this radiolabell will be incorporated in proteins which are already synthesized.
and during image analysis we will observe only radiolabelled proteins or all proteins.
can you send me the protocols which you used?
if i can do anything more in this please do tell me i would be happy to know your ideas.
bye
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