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Protein Liquid Chromatography (protein + liquid_chromatography)
Kinds of Protein Liquid Chromatography Selected AbstractsIgG2 containing IgM,IgG immune complexes predominate in normal human plasma, but not in plasma of patients with warm autoimmune haemolytic anaemiaEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 3 2006Dorothea Stahl Abstract:, The different physicochemical and sterical properties of IgG subclasses may favour a selective enrichment of defined IgG subclasses in IgM,IgG immune complexes (IC) of human plasma under physiological conditions. Such enrichment of IgG subclasses in IgM,IgG IC of plasma may differ from the normal IgG subclass distribution in plasma itself, and contribute to the physiological functions of IgM,IgG IC. Systematic studies on the IgG subclass distribution in IgM,IgG IC in humans are lacking. Using specific analytical techniques to characterise IgM,IgG IC in human plasma (i.e. fast protein liquid chromatography, enzyme-linked immunosorbent assay, affinity biosensor technology), and taking warm autoimmune haemolytic anaemia (WAIHA) of humans as a disease model, we here demonstrate that: (i) IgG2 is the predominant IgG subclass in IgM,IgG IC under physiological conditions, (ii) the predominance of IgG2 within IgM,IgG IC may get lost in polyclonal IgG-mediated autoimmune disease and (iii) the IgG subclass distribution in IgM,IgG IC influences the interaction between IC and blood cells involved in antigen presentation. The data presented here therefore extend the physiological function of IgG2, which is the protective immune response towards carbohydrate antigens in bacterial infections, and suggest IgG2-dependent regulation of immune responses to self-immunoglobulin in humans. The disturbed IgG subclass distribution in IgM,IgG IC of patients with WAIHA might influence activity of self-reactive B cells involved in the pathophysiology of the disease. [source] Isolation of DNA from genetically modified oils by fast protein liquid chromatographyINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 7 2010Li Huang Summary In this study, a novel method of fast protein liquid chromatography (FPLC) anion exchange chromatography was developed for isolation of DNA from processed genetically modified (GM) oils. Four kinds of different GM edible oil had been chosen as model sample. Salmon DNA was used as the control sample to determine the pH values and NaCl in mobile phase buffer. Applying pH 8 and NaCl gradient 0.5,2 m were chosen for the DNA isolation. The quality and purity of isolated DNA were tested with agarose gel electrophoresis, scanned with UV absorbance spectra and amplified by polymerase chain reaction (PCR). The result indicated that the quantity of DNA isolated by FPLC was suitable for further PCR analyses. Furthermore, it is more effective and less time-consuming in comparison with cetyltrimethylammonium bromide method and High Pure GMO Sample Preparation Kit method. [source] Partial purification and characterisation of banana [Musa (AAA Group) ,Gros Michel'] polyphenol oxidaseINTERNATIONAL JOURNAL OF FOOD SCIENCE & TECHNOLOGY, Issue 4 2009Chitsuda Chaisakdanugull Summary Polyphenol oxidase (PPO) from pulp of banana [Musa (AAA Group) ,Gros Michel'] was extracted and precipitated with 80% saturated ammonium sulphate followed by conventional column chromatography on Sephacryl S-200 HR and fast protein liquid chromatography on Mono Q column. The lyophilised PPO obtained from Sephacryl S-200 HR column was used for characterisation and inhibition studies. The partially purified PPO obtained from the Mono Q column exhibited at least three isoenzymes. The banana PPO had optimum pH for activity at 7 and it was stable around the same pH. Only 48% of initial enzyme activity was lost after heating at 70 °C for 30 min. The enzyme was completely inhibited by 2 mm sodium metabisulphite, 2 mm l -cysteine, 4 mm ascorbic acid, and 100 mm 4-hexylresorcinol. The Km and Vmax of banana PPO for dopamine were 2.08 mm and 0.124 mm min,1 respectively. [source] Increased sialylation of polymeric ,-IgA1 in patients with IgA nephropathyJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 1 2002Joseph C.K. Leung Abstract The mechanism of mesangial IgA deposition is poorly understood in IgA nephropathy (IgAN). Abnormal glycosylation of carbohydrate moieties in the hinge region of the IgA molecule has recently attracted much attention. In this report, we studied galactosylation and sialylation profiles in ,- and ,-IgA1 from patients with IgAN. Total serum IgA1 was isolated from patients with IgAN or healthy controls by jacalin-affinity chromatography. Six fractions of molecular weight (MW) 50,1,000 kDa were separated by fast protein liquid chromatography (FPLC). Four lectin-binding assays were used to study the sialylation and the presence of terminal galactose or N-acetylgalactosamine (GalNAc) in the O-linked carbohydrate moieties of ,- or ,-IgA1. Maackia amurensis agglutinin (MAA) and Sambucus nigra agglutinin (SNA) lectin recognize ,(2,3)- and ,(2,6)-linked sialic acid, respectively. Peanut agglutinin (PNA) and Helix aspersa (HA) lectin recognize terminal galactose and GalNAc, respectively. Reduced HA was demonstrated in macromolecular , or ,-IgA1 (300,825 kDa) isolated from patients with IgAN (P < 0.05 compared with healthy controls). Lambda- but not ,-IgA1 from patients with IgAN bound less to PNA (P < 0.05). The ,(2,3)-linked sialic acid content in ,- but not ,-IgA1 of MW 150,610 kDa from patients was higher than that of controls (P < 0.005). The ,(2,6)-linked sialic acid content in ,-IgA1 (300,825 kDa) and ,-IgA1 (150,610 kDa) from patients was also higher than that of controls. This unusual glycosylation and sialylation pattern of the ,-IgA1 may have important implications for the pathogenesis of IgAN, as both the masking effect of sialic acid on galactose and the reduced galactosylation will hinder the clearance of macromolecular ,-IgA1 by asialoglycoprotein receptor of hepatocytes. The negative charge from sialic acid may also favor mesangial deposition of macromolecular ,-IgA1 in IgAN. J. Clin. Lab. Anal. 16:11,19, 2002. © 2002 Wiley-Liss, Inc. [source] Production, purification and characterisation of a novel halostable xylanase from Bacillus sp.ANNALS OF APPLIED BIOLOGY, Issue 2 2010NTU-0 Bacillus sp. NTU-06 was used to produce xylanase, which is an important industrial enzyme used in the pulp and paper industry. The enzyme was purified by fast protein liquid chromatography (FPLC) and had a molecular mass of 24 kDa. The enzyme was active over a concentration range of 0,20% sodium chloride in culture broth, although its activity was optimal in 5% sodium chloride. A salinity stability test showed that 43% of the enzyme activity was retained after 4 h in 20% sodium chloride. Xylanase activity was maximal at pH 8.0 and 40°C. The enzyme was somewhat thermostable, retaining 20% of the original activity after incubation at 70°C for 4 h. The xylanase had Km and Vmax values of 3.45 mg mL,1 and 387.3 µmol min,1mg,1, respectively. The deduced internal amino acid sequence of Bacillus sp. NTU-06 xylanase resembled the sequence of beta-1,4-endoxylanase, which is a member of glycoside hydrolase family 11. Some of the novel characteristics that make this enzyme potentially effective in xylan biodegradation are discussed. [source] Purification of homogeneous forms of fungal peroxygenaseBIOTECHNOLOGY JOURNAL, Issue 11 2009René Ullrich Abstract Extracellular peroxygenase from the agaric fungus Agrocybe aegerita is a versatile biocatalyst that oxygenates various substrates by means of hydrogen peroxide. The enzyme is routinely produced in suspensions of soybean meal and has until now been purified by several steps of fast protein liquid chromatography (FPLC) using different ion exchangers. The final protein fraction had a molecular mass of 46 kDa but still consisted of several incompletely separated proteins with slightly differing isoelectric points (pI 5.2, 5.6, 6.1), probably representing differently glycosylated isoforms. This made it difficult to further purify the individual protein forms. Since homogeneous protein fractions are a pre-requisite for X-ray crystallography and specific structure-function studies, an appropriate FPLC procedure was developed starting with pre-purification of crude peroxygenase on SP Sepharose followed by chromatofocusing on a Mono P column and elution with a pH gradient. Three sufficiently separated main protein peaks were eluted from the Mono P column and confirmed to be distinct forms of aromatic peroxygenase with different pIs. All A. aegerita peroxygenase forms oxygenated toluene and naphthalene and no catalytic differences were observed between them. We tested also two devices for preparative isoelectric focusing (Rotofor, IsoPrime systems) for peroxygenase separation but resolution and protein recovery were not sufficient. [source] A low-molecular mass ribonuclease from the brown oyster mushroomCHEMICAL BIOLOGY & DRUG DESIGN, Issue 1 2005L. Xia Abstract:, A ribonuclease, with a molecular mass of 9 kDa and an N-terminal sequence resembling the sequence of a fragment of tRNA/rRNA cytosine-C5-methylase and a fragment of a alanyl-tRNA synthetase, was isolated from fresh fruiting bodies of the brown oyster mushroom Pleurotus ostreatus. The ribonuclease was purified using a very simple protocol that comprised ion-exchange chromatography on carboxymethyl (CM)-cellulose and affinity chromatography on Affi-gel blue gel. Subsequent gel filtration by fast protein liquid chromatography on Superdex 75 and sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis revealed that the ribonuclease was purified after the first two chromatographic steps. The ribonuclease was adsorbed on CM-cellulose and Affi-gel blue gel. The ribonuclease exhibited the highest activity toward poly A, lower activity toward poly C, slight activity toward poly G, and indiscernible activity toward poly U. The enzyme was stimulated upon exposure to 1 ,m Mg2+ and 10 ,m Zn2+, but was inhibited by the following ions at 10 mm: Ca2+, Mg2+, Zn2+, Cu2+, Fe2+, Mn2+, and Fe3+. The ribonuclease required a pH of 8.0 and a temperature of 50,70 °C to express maximal activity. It had a Km of 60 ,m toward yeast tRNA. It lacked mitogenic and HIV-1 reverse transcriptase inhibiting activities, but exerted antiproliferative activity toward leukemia L1210 cells. [source] | |||||||||||||