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protein fractionation

About microscopic forms of life, including Bacteria, Archea, protozoans, algae and fungi. Topics relating to viruses, viroids and prions also belong here.

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protein fractionation

Postby salmonela » Thu May 09, 2013 11:17 am

Hi all
I work on an uropathogenic strain of E.coli. When I artificially express from a plasmid one of the transcriptional regulator, it results in secretion of a protein factor in the media, which has interesting biological roles. My aim is to purify this unknown factor present in the growth media and to identify it by Mass Spec.
But the problem is I am not familiar with the fractionation methods. Looking through the literature, I could see that many different protocols are being employed out of which I don’t know which the best is. I like your suggestion with regard to a good standard protocol for identifying an unknown protein in the growth media.
Again to put it differently, I have protein in the supernatant of the growth media. I just need to identify it.
Thanks for all those who read this and Thank you very much for someone who comments.
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Postby JackBean » Thu May 09, 2013 2:03 pm

There are several ways how to purify your protein (bulk fractionation, liquid chromatography, electrophoresis). Best is if you have previously published protocol, at least of similar protein. If you have any, you just have to try different approaches and detect both your protein (enzymatic activity?, blotting...) and total protein and determine purification fold.
Great for you would be the first chapters from methods in Enzymology 463
http://www.sciencedirect.com/science/bo ... 766879/463
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.
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Postby drfuture » Mon May 20, 2013 3:34 pm

maybe 2-D polyacrylamide gel electrophoresis helpful for you, you can seperate, identify, and measure whole proteins in yur cell sample
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Postby JackBean » Mon May 20, 2013 7:03 pm

@drfuture - and how will salmonela know which one of the thousands proteins is her?
http://www.biolib.cz/en/main/

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Re:

Postby drfuture » Mon May 20, 2013 10:07 pm

JackBean wrote:@drfuture - and how will salmonela know which one of the thousands proteins is her?



yeah you are right, actually . i missed something, just as second year bachelors. sorry:(
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