crosslinking in organic buffers

Scott Craig s.craig at student.unsw.edu.au
Mon May 19 20:15:02 EST 2003


Hi All,
We are trying to couple a sulfhydryl labelled peptide to a lipid 
containing free amine groups. So far we have attempted to do this 
reaction in a two step process by reacting the NHS group of the LC-SMCC 
(a NHS and maleimide containing cross-linker) with the amines on the 
lipid in a buffer of DMSO+50uM triethylamine followed by reverse phase 
HPLC seperation and confirmation of the size using MS. This reaction 
appears to work from the MS results, however when we have subsequently 
tried to couple the maleimide group with the sulfhydryl group we appear 
to not get any coupling as analysed by HPLC. The maleimide/sulfhydryl 
labelling was done in 50% ethanol+50% HEPES+1mM EDTA. We have  used the 
LC-SMCC to fluorescently label a protein in aqueous buffers to confirm 
the reagent is working. Does anyone have any suggestions on how we could 
go about doing this crosslinking reaction in organic buffers or any 
other suggestions.
Thanks for any help.

-- 
Scott Craig
Biotechnology & Biomolecular Sciences
University of New South Wales
Sydney 2052, Australia






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