chend@bionette.ucs.orst.edu (Don Chen - Microbiology) (07/26/89)
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