Setting drops with proteins that are hard to concentrate

Dear Matt,

In one of our project, the protein of interest used to elute from Ni-column with
~20 other proteins. I also guessed it due to surface Cysteines.

Addition of 2mM free Cys in the protein sample helped, which competed with
free Cys on the protein surface and, ultimately, gave happy single band on
sds-page.

All the best
Manish

--
Center for Advanced Research in Biotechnology
University of Maryland Biotechnology Institute
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MD 20850 USA
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----- Original Message ----
From: "Bottomley, Matthew" <[log in to unmask]>
To: [log in to unmask]
Sent: Thursday, April 24, 2008 7:35:46 AM
Subject: [ccp4bb] Setting drops with proteins that are hard to concentrate

Setting drops with proteins that are hard to concentrate

Dear All,

I have a 50kDa protein that is soluble and monodisperse at up to approx 1mg/ml (after Ni-affinity and size-exclusion chromatography).

However, it aggregates (probably both via disulphides and via 'sticky/hydrophobic patches') when I concentrate it towards 2-3mg/ml, even in the presence of several detergents. I don't want to add DTT since my protein should have several intramolecular disulphides....although I do have 2 free Cysteines, partially exposed. I have already tried mutating the Cysteines, with little improvement.

Any suggestions for obtaining 5-10mg/ml?

Does anybody have good experiences with usin L-Arg and L-Glu (e.g. At 50mM) to aid concentrating (as in the Golovanov AP paper, JACS, 2004, pages 8933...)

Thanks for any input!

Yours,

Matt

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Matthew J. Bottomley, Ph.D.
Senior Research Biochemist
IRBM / MRL-Rome
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