CPS LPS EPS Structure
- Isolation of LPS – we use a variety of extraction methods to isolate crude LPS depending on nature of sample. For Smooth LPS and LOS we use: hot phenol/water, detergent, EDTA-TEA extraction. For rough LPS mutants, we employ phenol-chloroform-petroleum ether method (PCP). Extraction methods may need modifications based on specific nature of the project.
- Purification of LPS – crude LPS usually requires additional purification by ultracentrifugation and enzymatic digestion (nucleases, protease), de-lipidation, size exclusion chromatography (SEC) in associative and dissociative condition and progress of purification is verification by SDS-/DOC-PAGE.
- Analysis of O-chain and core region released from LPS – purification and size determination by FPLC size exclusion chromatography (SEC), high-performance anion exchange chromatography (HPAEC) and reverse phase chromatography; monosaccharide composition by GC-MS and GC-FID, GC-CI; glycosyl linkage of neutral and acidic sugars by method of partially methylated alditol acetates (PMAA); glycosyl linkage of of Kdo (2-keto-3-deoxy-octulosonic acid); determination of certain hexosaminuronic acids, deoxyheptulosaric acids, and pseudaminic acid; elucidation of glycosyl sequence and structure by 1D and 2D homo- and heteronuclear NMR, and high resolution MALDI-TOF MS and MS/MS, electrospray ionization (ESI-MS) mass spectrometry.
- Extraction and characterization of CPS – isolation and purification of capsular polysaccharides from Gram- negative and Gram-positive bacterial cell; monosaccharide and fatty acid composition by GC-MS and GC-FID, GC-CI; glycosyl linkage analysis by method of partially methylated alditol acetates (PMAA); purification and size determination by FPLC size exclusion chromatography (SEC), high-performance anion exchange chromatography (HPAEC); structure of polysaccharide and diacylglycerol lipid anchor moieties by MALDI-TOF MS and MS/MS, electrospray ionization (ESI-MS) and by 1D and 2D homo- and heteronuclear NMR.
- DOC-PAGE/ SDS-PAGE – polyacrylamide gel electrophoresis is used to demonstrate molecular size heterogeneity of intact LPS and CPS and track progress of purification and to provide evaluation of mutants defective in LPS/CPS biosynthesis in comparison with wild type strain and with reference lipopolysaccharides we could provide. Samples are stained with silver, alcian blue, zinc imidazole or the Molecular Probes ProQ Emerald stain.
- Wall teichoic and lipoteichoic acids (WTA/LTA) isolation and analysis – mechanical, chemical and enzymatic degradation of Gram-positive cells and chemical release and extraction of WTA and LTA from murein; composition analysis and structural characterization by high resolution mass spectrometry (MALDI-TOF MS and MS/MS).
- Exopolysaccharide (EPS) characterization – extracellular polysaccharides are isolated from bacterial culture media or from biofilm either in your laboratory or in our facility (please consult specific condition) and extensively purified by using size exclusion chromatography (SEC). EPS can be characterized by molecular size, monosaccharide composition, glycosyl linkage analysis by method of partially methylated alditol acetates (PMAA) and by 1D and 2D homo- and heteronuclear NMR; Comprehensive structural characterization often requires enzymatic depolymerization of EPS.