2D smear problem

Sebastian Bunka bunka at i112pc09.vu-wien.ac.at
Thu Apr 24 05:18:09 EST 1997


Hi netters,

We have a weird problem in 2D elektrophoresis.
We have cloned a bacterial outer membrane lipoprotein of an
Actinobacillus. When expressed the gene in E. coli and analysed in
Western blot (after denaturing SDS page) using convalescent phase 
sera (against the Actinob.) we find a strongly reactive band (or better
2 band close together, a) the "unprocessed", cytoplasmatic form, and
b) the "processed" (signal peptidase action and lipid modification...),
periplasmatic form.

OK, when now running a 2D gel (with all the "standard" buffers used
for routine 2D on serum samples, lysed lymphocytes ectc.) we get very 
nice gels when stained with silver. BUT after "Western"-blotting the
2D-gel we find a strong reactive "line" completely from the anode
to the cathode. MW is ok. Looks like someone had poured the protein
onto the slot AFTER the first dimension. But silver stain is OK ->
no "line" visible.

We also tried digestion of the sample w/ RNAse (DNAse not yet)
because someone mentioned complexes w/ DNA/RNA.

We are on the way to treat the samples w/ lipase (maybe the lipid 
modification causes the smearing.

Anyone observed things like this? Any hints how to treat the samples?
Any recommendation for a special kind of lipase?

Thanks in advance,
Sebastian
-- 

;-)
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Sebastian Bunka                 ph. (+43-1) 250 77 4208
Institute of                    FAX (+43-1) 250 77 4290
Medical Chemistry               email: Sebastian.Bunka at vu-wien.ac.at
University of Veterinary Medicine - Vienna - Austria
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