Dear Matt,
make sure that you don't have slow or even fast formation of unspecific
intermolecular disulfides by simple checks at several time points on
SDS-PAGE using sample buffer without DTT. Check the content of free
thiols and disulfides of your protein over a period of time. If you need
easy and solid protocols, just send me a mail.
Guenter
Bottomley, Matthew wrote:
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
====================================
Matthew J. Bottomley, Ph.D.
Senior Research Biochemist
IRBM / MRL-Rome
====================================
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