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Protein Purification Puzzle - Advice Needed!

Protein Purification Puzzle - Advice Needed! - Proteomics Forum

Protein Purification Puzzle - Advice Needed! - Post Questions and Discuss Proteomics, Proteomic Bioinformatics, Proteomic Techniques such as 2-D, Mass Spec etc.


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Old 02-27-2011, 11:10 PM
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Default Protein Purification Puzzle - Advice Needed!



Hi guys,

So I've been trying to purify a human protein for quite a while now with the ultimate aim of crystallising it. It's 25kDa, requires an MBP-His tag for soluble expression which I cleave with TEV and my plan is to separate the tag and protein with size exclusion. Unfortunately neither amylose nor nickel resins will pull this tag out completely.

The problem is that I get a good clean peak on my size exclusion trace but it turns out that it contains my cleaved MBP-His tag non-specifically bound to my protein. I looked to the postdocs for advice and they suggested using 10% glycerol during size exclusion to break up the complex. I just ran this (weekend science sucks) and it doesn't look like it changed anything at all

Has anybody around here been in and overcome such a situation in the past? I would be very, very grateful for any advice because I am running out of (realistic) ideas.


Also, I was wondering if anyone knows of any collections of case files for proteins. For example, information about the discovery of a protein, experiments that were used to define its function, methods which were used to purify it, problems that were encountered along the way and how they were solved. I want to get more protein experience under my belt but it's a slow process in the lab and I feel like I'm always taking advice from the senior people in the lab but not contributing much in return.

Thanks for any replies!
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Old 02-28-2011, 01:52 PM
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Default Re: Protein Purification Puzzle - Advice Needed!

His tags are charged when soluble. Using high concentrations of salt should help reduce that stickiness. Have you considered FPLC or HPLC?

Bryan
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