Path: utzoo!utgpu!watmath!iuvax!rutgers!orstcs!bionette.ucs.orst.edu!chend
From: chend@bionette.ucs.orst.edu (Don Chen - Microbiology)
Newsgroups: bionet.molbio.methds-reagnts
Subject: Quantitative assays of Lipopolysaccharide
Message-ID: <11837@orstcs.CS.ORST.EDU>
Date: 26 Jul 89 05:14:03 GMT
Sender: usenet@orstcs.CS.ORST.EDU
Reply-To: chend@bionette.ucs.orst.edu (Don Chen - Microbiology)
Distribution: bionet
Organization: Oregon State University - CMBL
Lines: 38
Keywords:lipopolysaccharide, assays, Vibrio anguillarum

I am presently working on a project involving the lipopolysaccharide
(LPS) of the marine pathogen _Vibrio__anguillarum.  I have used two
different protocols for extraction of the LPS.  One uses proteases
and the other uses acetone to remove free protein.  We have used the 
E. coli LPS as a positive control in qualitative assays.

I have run samples of the two VA-LPS extractions and the EC-LPS on
SDS-PAGE after boiling samples which include tracking dye and DTT.
After running the samples until the dye reaches the bottom of the
gel, I blot onto nitrocellulose or silver stain using periodate
instead of dichromate as the oxidant. I have also done a standard
Coomassie stain although this does not stain any of the LPS lanes.
We get banding patterns which -according to previously published
reports- identify the samples as coming from VA and EC.

My questions are these:

1. If there is little or no protein in the samples, what is being
stained?
2. If there is protein, how can I quantitate the amount of protein
that is there?
3. How do I quantitate the amount of polysaccharide versus the lipid
portion?
4. Assuming that I haptenate the VA-LPS with TNP, how do I measure
the amount of LPS in a sample of TNP-(VA)LPS? (That is, I have no
way of knowing the number of TNP molecules per LPS molecule that 
have been bound.)

I would appreciate any help. Thanks.

Don Chen
Dept of Microbiology
Oregon State University
Corvallis, OR 97331
(503) 737-3189

chend@bionette.cgrb.orst.edu
chend@beasley.ucs.orst.edu