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SDS-polyacrylamide Gel Electrophoresis (sds-polyacrylamide + gel_electrophoresis)
Selected AbstractsSodium dodecyl sulfate-capillary gel electrophoresis of polyethylene glycolylated interferon alphaELECTROPHORESIS, Issue 3 2004Dong H. Na Abstract Sodium dodecyl sulfate-capillary gel electrophoresis (SDS-CGE) using a hydrophilic replaceable polymer network matrix was applied to characterize the polyethylene glycol(PEG)ylated interferon alpha (PEG-IFN). The SDS-CGE method resulted in a clearer resolution in both the PEG-IFN species and the native IFN species. The distribution profile of PEGylation determined by SDS-CGE was consistent with that obtained by SDS-polyacrylamide gel electrophoresis (PAGE) with Coomassie blue or barium iodide staining. The result was also compared using matrix-assisted laser desorption/ionization-time of flight-mass spectrometry. SDS-CGE was also useful for monitoring the PEGylation reaction to optimize the reaction conditions, such as reaction molar ratio. This study shows the potential of SDS-CGE as a new method for characterizing the PEGylated proteins with advantages of speed, minimal sample consumption and high resolution. [source] Cloning and expression of Bacillus phytase gene (phy) in Escherichia coli and recovery of active enzyme from the inclusion bodiesJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008D.E.C.S. Rao Abstract Aims:, To isolate, clone and express a novel phytase gene (phy) from Bacillus sp. in Escherichia coli; to recover the active enzyme from inclusion bodies; and to characterize the recombinant phytase. Methods and Results:, The molecular weight of phytase was estimated as 40 kDa on SDS-polyacrylamide gel electrophoresis. A requirement of Ca2+ ions was found essential both for refolding and activity of the enzyme. Bacillus phytase exhibited a specific activity of 16 U mg,1 protein; it also revealed broad pH and temperature ranges of 5·0 to 8·0 and 25 to 70°C, respectively. The Km value of phytase for hydrolysis of sodium phytate has been determined as 0·392 mmol l,1. The activity of enzyme has been inhibited by EDTA. The enzyme exhibited ample thermostability upon exposure to high temperatures from 75 to 95°C. After 9 h of cultivation of transformed E. coli in the bioreactor, the cell biomass reached 26·81 g wet weight (ww) per l accounting for 4289 U enzyme activity compared with 1·978 g ww per l producing 256 U activity in shake-flask cultures. In silico analysis revealed a ,-propeller structure of phytase. Conclusions:, This is the first report of its kind on the purification and successful in vitro refolding of Bacillus phytase from the inclusion bodies formed in the transformed E. coli. Significance and Impact of the Study:, Efficient and reproducible protocols for cloning, expression, purification and in vitro refolding of Bacillus phytase enzyme from the transformed E. coli have been developed. The novel phytase, with broad pH and temperature range, renaturation ability and substrate specificity, appears promising as an ideal feed supplement. Identification of site between 179th amino acid leucine and 180th amino acid asparagine offers scope for insertion of small peptides/domains for production of chimeric genes without altering enzyme activity. [source] Activity-guided isolation of a novel protein from Croton tiglium with antifungal and antibacterial activitiesPHYTOTHERAPY RESEARCH, Issue 12 2008Muhammad Shahid Abstract This study describes the activity-guided isolation and purification of a novel antimicrobial protein from the seed of Croton tiglium Linn. Purification was carried out by (NH4)2SO4 precipitation, gel filtration and DEAE-cellulose ion-exchange chromatography. Antifungal and antibacterial activities were determined after each purification step. SDS-polyacrylamide gel electrophoresis revealed that the purified protein was a monomer with molecular mass of 50 kDa. This is a first report on purification of a protein from Croton tiglium, which possesses a strong and broad spectrum antimicrobial activity. Copyright © 2008 John Wiley & Sons, Ltd. [source] Problem-solving test: In vitro protein kinase a reaction,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2009József Szeberényi Terms to be familiar with before you start to solve the test. Protein kinases, protein phosphorylation, protein kinase A, tyrosine- and serine/threonine-specific kinases, extracellular signal-regulated kinases, Cyclin/Cdk complexes, SDS-polyacrylamide gel electrophoresis, Western blotting, diacylglycerol, adrenaline, autophosphorylation. [source] Problem-solving Test: ,1-antitrypsin deficiencyBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 4 2007An example for a protein folding disease Terms to be familiar with before you start to solve the test: protein conformation, protein folding, proteases, protein synthesis, protein glycosylation, glycoproteins, N-linked and O-linked oligosaccharides, endoplasmic reticulum, Golgi complex, secretory pathway, microsomes, pulse/chase labeling, SDS-polyacrylamide gel electrophoresis, immunoprecipitation, chaperones, protein translocation. [source] The effect of ,-amanitin on RNA polymerase II ubiquitination,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 5 2006Jozsef Szeberenyi Terms to be familiar with before you start to solve the test: RNA polymerase II, transcription, ,-amanitin, protein ubiquitination, synchronization of cell cultures, affinity adsorption, SDS-polyacrylamide gel electrophoresis, Western blotting, protein phosphorylation, protein kinases, in vitro transcription, plasmid, promoter, general and regulatory transcription factors, autoradiography. [source] Molecular cloning and immunoglobulin E reactivity of a natural rubber latex lecithinase homologue, the major allergenic component of Hev b 4CLINICAL & EXPERIMENTAL ALLERGY, Issue 11 2005E. Sunderasan Summary Background Hev b 4 is an allergenic natural rubber latex (NRL) protein complex that is reactive in skin prick tests and in vitro immunoassays. On SDS-polyacrylamide gel electrophoresis (SDS-PAGE), Hev b 4 is discerned predominantly at 53,55 kDa together with a 57 kDa minor component previously identified as a cyanogenic glucosidase. Of the 13 NRL allergens recognized by the International Union of Immunological Societies, the 53,55 kDa Hev b 4 major protein is the only candidate that lacks complete cDNA and protein sequence information. Objective We sought to clone the transcript encoding the Hev b 4 major protein, and characterize the native protein and its recombinant form in relation to IgE binding. Methods The 5,/3, rapid amplification of cDNA ends method was employed to obtain the complete cDNA of the Hev b 4 major protein. A recombinant form of the protein was over-expressed in Escherichia coli. The native Hev b 4 major protein was deglycosylated by trifluoromethane sulphonic acid. Western immunoblots of the native, deglycosylated and recombinant proteins were performed using both polyclonal antibodies and sera from latex-allergic patients. Results The cDNA encoding the Hev b 4 major protein was cloned. Its open reading frame matched lecithinases in the conserved domain database and contained 10 predicted glycosylation sites. Detection of glycans on the Hev b 4 lecithinase homologue confirmed it to be a glycoprotein. The deglycosylated lecithinase homologue was discerned at 40 kDa on SDS-PAGE, this being comparable to the 38.53 kDa mass predicted by its cDNA. Deglycosylation of the lecithinase homologue resulted in the loss of IgE recognition, although reactivity to polyclonal rabbit anti-Hev b 4 was retained. IgE from latex-allergic patients also failed to recognize the non-glycosylated E. coli recombinant lecithinase homologue. Conclusion The IgE epitopes of the Hev b 4 lecithinase homologue reside mainly in its carbohydrate moiety, which also account for the discrepancy between the observed molecular weight of the protein and the value calculated from its cDNA. [source] | |||||||||||||