need suggestions, protein isolation.

hnasko hnaskor at email.uc.edu
Mon Nov 29 10:59:14 EST 1999


November 29, 1999

I hope someone may have some suggestions.  I have isolated a protein >5000
MW <30,000 MW from cell conditioned media via RP-HPLC.  Sent the sample off
to a collaborator for sequence analysis and he cannot obtain data.  even
post trypsin digest the MS gives only peaks of 500-900 Da.  Based on the
chromatogram and the bioactivity of the sample I know it is there.  The
magnitude of the peak at 215 and 280 nm suggests there is enough to get
sequence 4x over.  We guess the problem is adsorption to the plastic
(untreated tube).  This is the most likly, i would hate to think they dont
know what they are doing.  Any suggestions as how we might get this protein
off the tube wall that is compatable with MS?  We are contemplating using a
detergent like SDS or TritonX followed by an HPLC step (scarry)?  We have no
structural info about this protein, only a bioactivity.  this tube
represents 6.5 liters of material and lots of work, I hate to think it is
lost.  We are going to restart purification with another 2 liters and are
thinking of ways to treat our tubes to avoid adsorption problems, ie
siliconization or teflon coated tubes.  I am not sure yet about the
compatability of the silicon with the MS (any problems there?)  Any
suggestions regarding these problems would be greatly appreciated.  Also if
you know a MS/protein core that is really good I am comtemplating trying a
different group for another sample and would appreciate a reference I could
contact.

thanx

Robert Hnasko
University of Cincinnati
College of Medicine
Dept. Cell biology, Neurobiology and Anatomy

email:  hnaskor at email.uc.edu










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