A simple procedure for labeling the lipid a moiety of lipopolysaccharide with I

A procedure for the iodination of lipid A is described. The iodinated product binds to rabbit spleen cells but not to red blood cells, and the binding to such spleen cells is inhibited by the presence of an excess of unlabeled lipid A. In dilute buffer solution, ¹²⁵I-labeled lipid A appears to be aggregated because it migrates on Sephadex G-100 in the region of the void volume, whereas it migrates as would a molecule even smaller than cytochrome c upon electrophoresis on polyacrylamide gel in the presence of sodium dodecyl sulfate. Evidence is presented that is consistent with the view that the iodine label is associated with the O-acyl groups of lipid A.     

A simple procedure for the preparation of protected 2''-O-methyl or 2''-O-ethyl ribonucleoside-3''-O-phosphoramidites.

Protected 2'-O-methyl and 2'-O-ethyl ribonucleoside-3'-O-phosphoramidites were prepared via alkylation of the ribonucleosides at an early stage in the synthesis. Utilizing a strategy of minimal protection, the alkylation was performed with unprotected cytidine and adenosine, or with O6-protected guanosine and N3,5'-O-protected uridine using methyl or ethyl iodide and sodium hydride. In subsequent steps, the introduction of standard protective groups for oligonucleotide synthesis and the concomitant separation from 3'-O-alkylated isomers was accomplished. A modification of the phosphitylation procedure permitted facile isolation of the desired phosphoramidites which show high coupling efficiencies in oligomer assembly.     

A simple procedure for the preparation and purification of the oligosaccharide components of the glycosylphosphatidylinositol anchor of membrane proteins

The oligosaccharide components of the glycosylphosphatidylinositol anchors of Trypanosoma brucei variant surface glycoproteins have been prepared and purified by treatment with hydrolytic enzymes and solvent extraction procedures followed by HPLC purification using a specific oligosaccharide binding matrix (Glyco-Pak N, by Waters). Three oligosaccharide peaks (peaks I, II and III) were resolved by a single isocratic HPLC step (70% acetonitrile in water). The material from these peaks was hydrolyzed in acid and analyzed by GC/MS. GC/MS analysis of the material obtained from each peak demonstrated the presence of inositol, glucosamine, and mannose in a 1:1:3 ratio. A variable number of galactose residues were detected in each peak. The galactose:inositol ratios of the purified components were 1:1, 2:1, and 3:1 for peaks I, II and III, respectively, suggesting that the separation obtained depends primarily on the number of sugar residues present in each fraction.     

A simple procedure for the purification of rat alpha-fetoprotein

A simple purification procedure for obtaining a high yield of electrophoretically and immunologically pure rat α-fetoprotein from amniotic fluid is described. Rat amniotic fluid is passed through an anti-rat albumin immunoabsorbent column to remove albumin. The albumin-free eluate is then chromatographed on DEAE-Sephacel to separate α-fetoprotein from transferrin and other minor protein contaminants. This two-step purification procedure results in a recovery of approximately 70% of the rat α-fetoprotein originally present in the amniotic fluid.     

A simple procedure for large-scale preparation of pure plasmid DNA free from chromosomal DNA from bacteria

A very simple, inexpensive procedure for preparing pure plasmid DNA from bacteria is described. In this method, lysozyme-induced spheroplasts are made in presence of 833 micrograms/ml of ethidium bromide which are then lysed by a mixture of Brij 58 and sodium deoxycholate, and the lysate is centrifuged at 48,000 g for 25 min whereby about 99.9% of total chromosomal DNA is pelleted. From the supernatant containing plasmid DNA, the proteins are removed by phenol extraction and the major part of RNA by CaCl2 precipitation, and finally the small amount of residual RNA is removed by RNase treatment. The average yield of pBR322 DNA from 1 liter of amplified culture by this procedure is 2 to 2.5 mg and the preparation is highly pure, containing only about 0.005% of total yield as chromosomal DNA contaminant. Moreover, the substrate activity and the transforming ability of the plasmid DNA prepared by this method remain unaffected.     

A simple assay procedure for beta-D-mannanase.

A simple assay procedure for beta-D-mannanase enzyme has been developed which employs carob D-galacto-D-mannan dyed with Remazolbrilliant Blue. Additionally, the procedure is quantitative, relatively sensitive, and highly specific for beta-D-mannanase enzyme. It can be readily used for the determination of beta-D-mannanase activity in crude enzyme preparations and column-chromatography eluates.     

A simple procedure for cross-linking complementary oligonucleotides

A simple, efficient procedure for cross-linking two complementary oligonucleotides, which does not require chemical modification of either oligonucleotide, is described. One of the oligonucleotides is first converted to the 5'-phosphorothioate derivative with polynucleotide kinase. It is then incubated with its complement in the presence of 1 microM trans-platinum(II)diammine dichloride. After overnight incubation, 40-50% cross-linking is observed. DNA synthesis by the Klenow fragment of Escherichia coli DNA polymerase I is blocked at the cross-linked site, resulting in the formation of truncated products. Potassium platinous chloride (K2PtCl4) and cis-platinum(II)diammine dichloride form cross-links less efficiently than the trans isomer.