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Tara K. Sigdel - One of the best experts on this subject based on the ideXlab platform.
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Fractionation of Soluble Proteins Using DEAE-Sepharose, SP-Sepharose, and Phenyl Sepharose Chromatographies for Proteomics.
Methods in molecular biology (Clifton N.J.), 2017Co-Authors: Parhom Towfighi, Jacob S. Shaw, Tara K. SigdelAbstract:In order to simplify a complex mixture of soluble proteins from tissues, a protocol to fractionate samples prior to two-dimensional (2D) gel electrophoresis has been developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins with DEAE-Sepharose can result in an increase in the number of detectable 2D gel spots. These gel spots are amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. The DEAE-Sepharose column fractionation acts to partition soluble proteins from cell extracts. Similarly, a SP-Sepharose column can fractionate soluble proteins and increase the number of detectable gel spots. Lastly, fractionation of cell extract with a phenyl Sepharose column can also result in an increase in the number of detectable 2D gel spots. This chapter describes an easy, inexpensive way to fractionate soluble proteins and a way to better profile proteomes.
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Fractionation of soluble proteins in Escherichia coli using DEAE-, SP-, and phenyl sepharose chromatographies.
Journal of biomolecular techniques : JBT, 2004Co-Authors: Tara K. Sigdel, Renee Cilliers, Priya R. Gursahaney, Michael W. CrowderAbstract:In an effort to simplify a complex mixture of soluble proteins from Escherichia coli, methods to fractionate the samples prior to two-dimensional (2D) gel electrophoresis were developed. These methods involve the use of DEAE-Sepharose, SP-Sepharose, and phenyl Sepharose chromatographic columns and the fractionation of the protein mixtures based on differential anionic, cationic, and hydrophobic properties of the proteins, respectively. Fractionation of the soluble proteins from an E. coli extract with DEAE-Sepharose resulted in a threefold increase in the number of detectable 2D gel spots. These gel spots were amenable to protein identification by using in-gel trypsin digestions, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and peptide mass fingerprinting. Significantly, the DEAE-Sepharose column fractionation effectively partitioned the soluble proteins from the cell extracts. Similarly, an SP-Sepharose column was used to fractionate the soluble proteins from E. coli and resulted in over a twofold increase in the number of detectable gel spots. Lastly, fractionation of the cell extract with the phenyl Sepharose column resulted in a threefold increase in the number of detectable 2D gel spots. This work describes an easy, inexpensive way to fractionate the soluble proteins in E. coli and a way to better profile the E. coli proteome.
Renee Norberg - One of the best experts on this subject based on the ideXlab platform.
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preparation of highly purified f actin depolymerizing factor of human serum
FEBS Journal, 2008Co-Authors: Rigmor Thorstensson, Catharina Sterky, Renee NorbergAbstract:F-actin depolymerizing factor (ADF) of human serum was purified 250–400-fold to more than 98% purity with high reproducibility. The purification included (1) 30–50% (NH4)2SO4 precipitation, (2) ion-exchange chromatography on DEAE-Sepharose, (3) chromatofocusing on Polybuffer exchanger 94 and (4) affinity chromatography on ConA-Sepharose. The recovery of ADF was estimated to be 20–30% whereas the ADF activity yield was 5–17%. The lower activity yield was thought to be due-partly to proteolysis and partly to destabilization of highly purified ADF.
D. Maher - One of the best experts on this subject based on the ideXlab platform.
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Prion-removal capacity of chromatographic and ethanol precipitation steps used in the production of albumin and immunoglobulins.
Vox sanguinis, 2006Co-Authors: J. Thyer, A. Unal, P. Thomas, B. Eaton, R. Bhashyam, J. Ortenburg, E. Uren, Deborah Middleton, P. Selleck, D. MaherAbstract:Background and Objectives Although there is no epidemiological evidence to suggest that classical Creutzfeldt–Jakob disease (CJD) is transmitted through blood or blood products, the variant form (vCJD) has been implicated in transmission via packed red blood cells. The potential threat of the infectious agent contaminating plasma pools has led to manufacturing processes being examined for capacity to remove prions. The objective of these studies was to examine the prion-removal potential of the chromatographic purification and ethanol precipitation steps used to fractionate immunoglobulins and albumin from human plasma. Materials and Methods Western blot assay was used to examine the partitioning of proteinase K-resistant scrapie prion protein (PrPsc) over DEAE Sepharose, CM Sepharose and Macro-Prep High Q chromatographic columns, utilizing microsomal scrapie 263K spiked into each scaled down feedstream and assayed after each chromatographic step. In further studies, bioassay in C57 black mice was used and spikes of 10 000 g clarified brain homogenate of scrapie ME7 were added to feedstreams before sequences of scaled down chromatographic or Cohn fractionation process steps. Results The microsomal spiking study with Western blot detection demonstrated substantial partitioning of PrPsc away from the target proteins in all ion exchange chromatographic steps examined. The log10 reduction factors (LRF) across DEAE Sepharose and CM Sepharose columns for albumin were ≥ 4.0 and ≥ 3.0 respectively. The reductions across DEAE Sepharose and Macro-Prep High Q for intravenous immunoglobulin were 3.3 and ≥ 4.1 respectively. Bioassay demonstrated LRFs of ≥ 5.6 across the combination of DEAE Sepharose and CM Sepharose columns in the albumin process and ≥ 5.4 across the combination of DEAE Sepharose and Macro-Prep High Q columns in the intravenous immunoglobulin process. Bioassay studies also demonstrated a LRF of ≥ 5.6 for immunoglobulin produced by Cohn fractionation. Conclusions Using rodent-adapted scrapie as a model, the studies indicated that ion exchange chromatography, as well as Cohn immunoglobulin fractionation have the potential to effectively reduce the load of TSE agents should they be present in plasma pools. Table of Contents Ion exchange columns used for production of human albumin and immunoglobulins, as well as Cohn immunoglobulin fractionation, effectively reduce the load of TSE agents should they be present in plasma pools.
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Investigation of prion removal/inactivation from chromatographic gel.
Vox sanguinis, 2006Co-Authors: J. Thyer, A. Unal, E. Uren, Deborah Middleton, Gunter Hartel, J. Bingham, M. Braun, D. MaherAbstract:Background and Objectives Concerns about the potential for prions to be retained on chromatography gels during the manufacture of plasma products prompted development of an investigational strategy for detecting infectious prions bound to gels. The objective was to firstly examine methods of implanting gels intracerebrally (IC) in mice, then to examine prion cleaning from a scaled-down version of the DEAE Sepharose column used in a production process to fractionate immunoglobulins and albumin from human plasma. Materials and Methods The study consisted of two parts: (i) the pathophysiological impact by IC inoculation of ground gel beads was compared to whole gel beads; (ii) the feedstreams to two DEAE Sepharose columns were spiked with scrapie ME7. One column was subjected to the protein loading and elution portions of the chromatography cycle. The other column was subjected to the full cycle of protein loading and elution, followed by regeneration with 0·5 m NaCl, 1 m NaOH and solvent/detergent washes. The gels were unpacked and bioassayed by IC implantation in mice to quantify infectivity. Results IC inoculation of ground gel beads resulted in unacceptably high pathological impact in the mice whereas whole gel bead inoculation resulted in a reduced affect. Accordingly, the whole bead model system was used to assess prion removal/inactivation from chromatography gels at the pre- and postcleaning stage of the chromatography cycle. Infectious prions were detected on the DEAE Sepharose prior to the cleaning step; however, the gel cleaning cycle reduced infectivity by a log reduction factor (LRF) of ≥ 2·75, thus reducing infectivity by bioassay to below detectable limits. Conclusions A model system for assessment of prion inactivation/removal from chromatography gels has been established. Spiked prion infectivity does bind to DEAE Sepharose gel; however, the cleaning cycle removed infectivity to levels below that detectable by bioassay.
Jiansong Cheng - One of the best experts on this subject based on the ideXlab platform.
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Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides.
Analytical and bioanalytical chemistry, 2016Co-Authors: He Zhu, Cong Xiao, Kuan Jiang, Ebtesam A Gashash, Ding Liu, Jing Song, Jiansong ChengAbstract:N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment. This method integrated the advantages of Click Maltose and zwitterionic HILIC (ZIC-HILIC) and showed a relatively higher specificity for N-glycosylated peptides. This strategy was then applied to tryptic digests of normal human serum, followed by deglycosylation using peptide-N-glycosidase F (PNGase F) in H2 18O. Subsequent LC–MS/MS analysis allowed for the assignment of 219 N-glycosylation sites from 115 serum N-glycoproteins. This study provides an alternative approach for N-glycopeptide enrichment and the method employed is effective for large-scale N-glycosylation site identification.
Rigmor Thorstensson - One of the best experts on this subject based on the ideXlab platform.
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preparation of highly purified f actin depolymerizing factor of human serum
FEBS Journal, 2008Co-Authors: Rigmor Thorstensson, Catharina Sterky, Renee NorbergAbstract:F-actin depolymerizing factor (ADF) of human serum was purified 250–400-fold to more than 98% purity with high reproducibility. The purification included (1) 30–50% (NH4)2SO4 precipitation, (2) ion-exchange chromatography on DEAE-Sepharose, (3) chromatofocusing on Polybuffer exchanger 94 and (4) affinity chromatography on ConA-Sepharose. The recovery of ADF was estimated to be 20–30% whereas the ADF activity yield was 5–17%. The lower activity yield was thought to be due-partly to proteolysis and partly to destabilization of highly purified ADF.