Purification of LPS Binding Factors in Tolerant Serum by Affinity Chromatography
Abstract
Towards the end of the last period we radio labeled E. coli Olll in the LPS component using the gal E mutant of this strain in media containing tritiated galactose. We then used the hot phenol method to extract the radio labeled LPS. Our yield was 2.53 mg of LPS with a specific activity of 15800 cpm/mg. We will use this reagent in the above experiments. Since the last report we have isolated the lipoproteins from 10 mls of normal and tolerant sera by ultracentrifugation and prepared frozen 1.0 ml aliquots which contain activity. We passed these over a 1 meter sepharose 6B column and also over a Superose 6 HPLC column. We found that while the elution profiles of the lipoproteins from normal and tolerant sera were different, that activity in our radioimmunoassay was lost in passage over each column. As in the experiments described above, we believe that a difficulty with the large column is that we may be losing activity in the process of concentrating the fractions. As noted in our original proposal, losses by absorption to glass and plastic ware are likely to be high because of the hydrophobicity of the lipoproteins. We are now concentrating on the HPLC which has a higher resolution and which permits the repetition of many identical runs.
Document Details
- Document Type
- Technical Report
- Publication Date
- Mar 12, 1991
- Accession Number
- ADA233638
Entities
People
- H. S. Warren
Organizations
- Massachusetts General Hospital