glutathione sepharose 4 fast flow (GE Healthcare)
99
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Name:
Glutathione sepharose 4B
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Catalog Number:
GS2376858
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85
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Structured Review
GE Healthcare
glutathione sepharose 4 fast flow
https://www.bioz.com/result/glutathione sepharose 4 fast flow/product/GE Healthcare
Average 99 stars, based on 84 article reviews
Price from $9.99 to $1999.99
https://www.bioz.com/result/glutathione sepharose 4 fast flow/product/GE Healthcare
Average 99 stars, based on 84 article reviews
Price from $9.99 to $1999.99
glutathione sepharose 4 fast flow - by Bioz Stars,
2020-01
99/100 stars
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Images
1) Product Images from "Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding"
Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding
Journal:
doi: 10.1194/jlr.M041129
Figure Legend Snippet: Binding of PCSK9 to recombinant EGF-A. A: Purification of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow affinity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose
Techniques Used: Binding Assay, Recombinant, Purification, Flow Cytometry, Chromatography, Size-exclusion Chromatography
2) Product Images from "Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication"
Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication
Journal: PLoS Pathogens
doi: 10.1371/journal.ppat.1006851
Figure Legend Snippet: NP interacts with PLSCR1 in mammalian cells. (A, B) Co-IP assay of V5-NP and Flag-PLSCR1 in HEK293T cells. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates were immunoprecipitated with a mouse anti-V5 mAb (A) or a mouse anti-Flag mAb (B) and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (C, D) GST pull-down assay of NP and PLSCR1. Lysates of HEK293T cells transfected with the GST or GST-PLSCR1 construct were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-WSNNP (C); HEK293T cell lysates containing exogenously expressed GST or GST-WSNNP were incubated with Glutathione Sepharose 4 Fast Flow and then mixed with lysates from cells transfected with pCAGGS or pCAGGS-PLSCR1 (D). After washing away the unbound proteins, equal volumes of proteins bound to the beads and the original cell lysates (5% of the input) were examined by western blotting using a rabbit anti-NP pAb, a rabbit anti-GST pAb, or a rabbit anti-PLSCR1 pAb. GST, GST-PLSCR1, or GST-WSNNP proteins in the eluates were detected by Coomassie blue (CB) staining. (E) The NP-PLSCR1 interaction does not rely on RNA binding. HEK293T cells were transfected individually or in combination with plasmids expressing V5-WSNNP and Flag-PLSCR1. Cell lysates treated with RNase A/T1 or left untreated were immunoprecipitated with a mouse anti-Flag mAb and were subjected to western blotting with a rabbit anti-V5 pAb or a rabbit anti-Flag pAb to reveal the presence of NP and PLSCR1, respectively. (F) PLSCR1 interacts with NP during natural viral infection. Confluent A549 cells grown in 6-well plates were mock infected with PBS or infected with WSN virus at an MOI of 5. At 6 h p.i., cell lysates were immunoprecipitated with a rabbit anti-PLSCR1 pAb and were subjected to western blotting with a mouse anti-NP mAb or a rabbit anti-PLSCR1 pAb to detect NP and PLSCR1, respectively. (G) Mapping of the PLSCR1-interacting domain within NP. Schematic presentation of influenza NP showing the different domains as well as the truncation mutants made in this study is on the left side. The interaction between PLSCR1 and the NP truncation mutants is shown on the right side. Lysates of HEK293T cells were pulled down with glutathione magnetic beads. The bound proteins were subjected to western blotting with a rabbit anti-Flag pAb or a rabbit anti-GST pAb to reveal the presence of PLSCR1 and NP, respectively. NES, nuclear export signal; NAS, nuclear accumulation signal.
Techniques Used: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads
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Clone Assay:Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: Immunoblots were visualized by enhanced chemiluminescence method. .. GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair Article Snippet: The coding sequences of TDG, Dnmt3a were cloned into pGEX4T-3 (Pharmacia) for expression of the GST-fusions in the E.coli strain BL21 (DE3). .. Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using Centrifugation:Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Protein extracts were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge 5415D for 10 min at 4°C, mixed with anti-FLAG M2 agarose (Sigma-Aldrich) and incubated for 2 hr at 4°C. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: When the optical density reached 1.0, they were transferred to the low temperature (16 °C) shaker, and induced with 0.25 mM isopropyl-D-thiogalactoside (IPTG) for 16 h. After that, cells were harvested and then resuspended in lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, 500 mM NaCl, 2 mM MgCl2 , 5% glycerol for GST-tagged fusion proteins) supplemented with 1 mM PMSF. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation Article Snippet: For the GST pull-down assays equal amounts of fusion proteins were incubated with a defined quantity of cell lysate. .. The proteins bound to the Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: The bacterial cultures were centrifuged, and the resulting pellets were resuspended in 500 μl of GST-lysis buffer (1 m m EDTA, 0.5% Nonidet P-40) and protease inhibitors in standard phosphate-buffered saline (PBS). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions Article Snippet: In vitro pull-down experiments : Protein samples were mixed in their correct stoichiometric ratios, using 10 µM She2p wt/ Δhelix E, 10 µM She3p-His6 and 5 µM GST-Myo4-C in a final volume of 100 µl pull-down buffer (20 mM Hepes pH 7.8, 140 mM or 200 mM NaCl, 2 mM MgCl2 , 2 mM DTT). .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Article Title: Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: Cells were harvested by centrifugation, suspended in 1 m M EDTA, 5 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 µ M leupeptin, 1 µ M pepstatin A, 0.1 mg ml−1 lysozyme, Dulbecco’s phosphate-buffered saline that contained neither calcium chloride nor magnesium chloride (PBS; Sigma–Aldrich, St Louis, Missouri, USA) and sonicated and the crude lysate was centrifuged. .. The supernatant was applied onto a 50 ml Autoradiography:Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Construct:Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Inserts were sequenced to confirm their fidelity, and then the plasmid constructs were transformed into the Epicurian Coli® BL21-CodonPlus™ strain (Stratagene, Cedar Creek, TX). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A Article Snippet: pET29a-Aurora-A and pGEX4T2-PUM2 constructs were tramsformed in E. Coli BL21 (DE3). .. Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Ubiquitin Assay:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: PPARγ2-ubiquitin conjugates were prepared by an in vitro ubiquitination assay. .. GST pull-down assays were performed according to the instructions of the manufacturer ( Incubation:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B Article Snippet: TFIIS-GST was purified by affinity chromatography on Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The obtained solution was incubated in the presence of 1% Triton X-100 for 1 h at 4°C. .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Protein extracts were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge 5415D for 10 min at 4°C, mixed with anti-FLAG M2 agarose (Sigma-Aldrich) and incubated for 2 hr at 4°C. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: When the optical density reached 1.0, they were transferred to the low temperature (16 °C) shaker, and induced with 0.25 mM isopropyl-D-thiogalactoside (IPTG) for 16 h. After that, cells were harvested and then resuspended in lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, 500 mM NaCl, 2 mM MgCl2 , 5% glycerol for GST-tagged fusion proteins) supplemented with 1 mM PMSF. .. 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Proteins were then purified by incubating cell lysates with a reduced form of Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions Article Snippet: GST-Nef, sdAb19, and Neffins were produced in Escherichia coli strain BL21 as described previously ( , ). .. Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions Article Snippet: In vitro pull-down experiments : Protein samples were mixed in their correct stoichiometric ratios, using 10 µM She2p wt/ Δhelix E, 10 µM She3p-His6 and 5 µM GST-Myo4-C in a final volume of 100 µl pull-down buffer (20 mM Hepes pH 7.8, 140 mM or 200 mM NaCl, 2 mM MgCl2 , 2 mM DTT). .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A Article Snippet: Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Article Title: Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: The cells, which were suspended in 4.8 l LeMaster medium (LeMaster & Richards, 1985 ) supplemented with 25 µg ml−1 SeMet and 100 µg ml−1 ampicillin and grown to an OD of 0.5–0.8, were incubated at 303 K for 4 h after protein expression had been induced with 1 m M IPTG (final concentration). .. The supernatant was applied onto a 50 ml Expressing:Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: Paragraph title: Expression and purification of AHSPWT /α-Hb complexes ... The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: For purification of FLAG-tagged proteins from S. pombe cells, cells expressing FLAG-tagged proteins were cultured in YES medium and collected when an optical density of 1.2 at 600 nm was reached. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair Article Snippet: Paragraph title: Protein expression and purification ... Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation Article Snippet: Paragraph title: Expression, purification of recombinant fusion proteins and GST pull-down ... The proteins bound to the Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer Article Snippet: Vectors expressing GST fused to full-length p23 (GST-p23); its N terminus (GST-NTD) or empty vector (pGEX-6P-1) was expressed in BL21-codon plus Escherichia coli . .. The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Protein expression was then induced with the addition of 0.4 m m isopropyl 1-thio-β-galactopyranoside, and cultures were incubated a further 2.5 h at 30 °C. .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: Expression was induced with 0.5 mM isopropyl β-d -thiogalactopyranoside for 5 h at 25°C. .. The fusion protein was purified by affinity chromatography on Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: Paragraph title: 2.1. Protein expression and purification ... The supernatant was applied onto a 50 ml Modification:Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch Article Snippet: Sc787 cells cotransformed with plasmids carrying GST- GAL3 and GAL80 were grown to midlog phase and whole-cell extracts containing GST-Gal3 and Gal80 were prepared as described , using a modified lysis buffer (20 m m HEPES pH 7.4, 0.5% Triton X-100, 200 m m NaCl, 0.5 m m EDTA, 2 m m DTT, and 5 m m MgCl2 ). .. Western Blot:Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: Paragraph title: Immunoprecipitation and Western blot analysis ... GST-GULP was pulled down using Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions Article Snippet: Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch Article Snippet: Transformation Assay:Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: Briefly, the plasmid was transformed into BL21 (DE3) cells and grown in Terrific Broth at 37 °C. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Inserts were sequenced to confirm their fidelity, and then the plasmid constructs were transformed into the Epicurian Coli® BL21-CodonPlus™ strain (Stratagene, Cedar Creek, TX). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: Sequences encoding full-length Sec10 or its amino-terminal region (Sec10NTer; aa 378–708) were subcloned into pGEX4T1 at the carboxy terminus of GST and transformed in E. coli BL21(DE3) strain. .. The fusion protein was purified by affinity chromatography on Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: The resulting plasmid was transformed into Escherichia coli B834(DE3) (Novagen, Madison, Wisconsin, USA). .. The supernatant was applied onto a 50 ml Transfection:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or HA-TRIM23 were harvested and lysed. .. GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: Transfected cells were harvested in 500 μl of NP-40 lysis buffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1% NP-40, 10% glycerol, 1 mM Na3 VO4 , and Complete Mini EDTA-free Protease Inhibitor Cocktail tablet [Roche, Indianapolis, IN]). .. GST-GULP was pulled down using Concentration Assay:Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: The cells, which were suspended in 4.8 l LeMaster medium (LeMaster & Richards, 1985 ) supplemented with 25 µg ml−1 SeMet and 100 µg ml−1 ampicillin and grown to an OD of 0.5–0.8, were incubated at 303 K for 4 h after protein expression had been induced with 1 m M IPTG (final concentration). .. The supernatant was applied onto a 50 ml Protease Inhibitor:Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Cells were then lysed with glass beads in lysis buffer A {50 mM Tris-HCl (ph 8.0), 150 mM NaCl, 0.1% NP-40, 10% glycerol, 50 mM NaF, 1 mM Na3 VO4 , 5 mM EDTA, 5 mM N -methylmaleimide, 1 µM microcyctin, 0.1 µM okadaic acid, 0.2 mM p -4-amidoinophenyl-methane sulfonyl fluoride hydrochloride monohydrate (p -APMSF) and Roche complete EDTA-free protease inhibitor cocktail} using a FastPrep cell disrupter (Qbiogene) for two cycles of 20 seconds each at speed 6, with a one-minute interval on ice between the two cycles. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: Transfected cells were harvested in 500 μl of NP-40 lysis buffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1% NP-40, 10% glycerol, 1 mM Na3 VO4 , and Complete Mini EDTA-free Protease Inhibitor Cocktail tablet [Roche, Indianapolis, IN]). .. GST-GULP was pulled down using Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch Article Snippet: Protease inhibitor cocktails (PIC) (PIC-D, 88 mg/ml PMSF and 1 mg/ml pepstatin A in DMSO; PIC-W, 157 mg/ml benzamidine, 0.5 mg leupeptin, and 0.5 mg bestatin in water) were added to all lysis buffer and all subsequent solutions at 1/1000 dilutions. .. Cell Culture:Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: For purification of FLAG-tagged proteins from S. pombe cells, cells expressing FLAG-tagged proteins were cultured in YES medium and collected when an optical density of 1.2 at 600 nm was reached. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to Hemagglutination Assay:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: HEK293T cells transiently transfected with plasmids encoding FLAG-PPARγ2 and/or HA-TRIM23 were harvested and lysed. .. GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: GST-GULP was pulled down using Polymerase Chain Reaction:Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: These PCR products were subcloned in-frame into the bacterial expression vector pGEX-2T (Amersham Biosciences). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Sonication:Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: The bacterial cultures were centrifuged, and the resulting pellets were resuspended in 500 μl of GST-lysis buffer (1 m m EDTA, 0.5% Nonidet P-40) and protease inhibitors in standard phosphate-buffered saline (PBS). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: Cells were harvested by centrifugation, suspended in 1 m M EDTA, 5 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 µ M leupeptin, 1 µ M pepstatin A, 0.1 mg ml−1 lysozyme, Dulbecco’s phosphate-buffered saline that contained neither calcium chloride nor magnesium chloride (PBS; Sigma–Aldrich, St Louis, Missouri, USA) and sonicated and the crude lysate was centrifuged. .. The supernatant was applied onto a 50 ml Recombinant:Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation Article Snippet: Paragraph title: Expression, purification of recombinant fusion proteins and GST pull-down ... The proteins bound to the Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions Article Snippet: GST-Nef, sdAb19, and Neffins were produced in Escherichia coli strain BL21 as described previously ( , ). .. Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A Article Snippet: pET29a-Aurora-A and pGEX4T2-PUM2 constructs were tramsformed in E. Coli BL21 (DE3). .. Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: The fusion protein was purified by affinity chromatography on Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: Paragraph title: Purification of recombinant proteins expressed in E. coli ... Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using GST Pulldown Assay:Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions Article Snippet: Paragraph title: GST pulldown assay. ... Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of Pull Down Assay:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: Paragraph title: GST pull-down assay ... GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: Paragraph title: Protein purification and GST pull-down assay ... Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer Article Snippet: Paragraph title: GST pull-down assay ... The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch Article Snippet: Paragraph title: Pull-down assay for GST-Gal3 and Gal80 interaction: ... Methylation:Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Mutagenesis:Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: The mutations Thr431Lys, Gln432Leu, Thr451Ala and Ala624Gly were then introduced using a QuikChange Site-Directed Mutagenesis Kit (Stratagene, La Jolla, California, USA). .. The supernatant was applied onto a 50 ml Subcloning:Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Flow Cytometry:Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: When the optical density reached 1.0, they were transferred to the low temperature (16 °C) shaker, and induced with 0.25 mM isopropyl-D-thiogalactoside (IPTG) for 16 h. After that, cells were harvested and then resuspended in lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, 500 mM NaCl, 2 mM MgCl2 , 5% glycerol for GST-tagged fusion proteins) supplemented with 1 mM PMSF. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: Cells were harvested by centrifugation, suspended in 1 m M EDTA, 5 m M 2-mercaptoethanol, 1 m M phenylmethylsulfonyl fluoride (PMSF), 1 µ M leupeptin, 1 µ M pepstatin A, 0.1 mg ml−1 lysozyme, Dulbecco’s phosphate-buffered saline that contained neither calcium chloride nor magnesium chloride (PBS; Sigma–Aldrich, St Louis, Missouri, USA) and sonicated and the crude lysate was centrifuged. .. The supernatant was applied onto a 50 ml Purification:Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B Article Snippet: For IL-6 induction, the cells were serum-starved for at least 16 h, and IL-6 was added at a final concentration of 10 ng/ml for 30 min. .. TFIIS-GST was purified by affinity chromatography on Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: Immunoblots were visualized by enhanced chemiluminescence method. .. GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The obtained solution was incubated in the presence of 1% Triton X-100 for 1 h at 4°C. .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Protein extracts were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge 5415D for 10 min at 4°C, mixed with anti-FLAG M2 agarose (Sigma-Aldrich) and incubated for 2 hr at 4°C. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair Article Snippet: Paragraph title: Protein expression and purification ... Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation Article Snippet: Paragraph title: Expression, purification of recombinant fusion proteins and GST pull-down ... The proteins bound to the Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer Article Snippet: Vectors expressing GST fused to full-length p23 (GST-p23); its N terminus (GST-NTD) or empty vector (pGEX-6P-1) was expressed in BL21-codon plus Escherichia coli . .. The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: The bacterial cultures were centrifuged, and the resulting pellets were resuspended in 500 μl of GST-lysis buffer (1 m m EDTA, 0.5% Nonidet P-40) and protease inhibitors in standard phosphate-buffered saline (PBS). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions Article Snippet: She2p, She2p (Δhelix E), and She3p were expressed and purified as previously described. .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: Expression was induced with 0.5 mM isopropyl β-d -thiogalactopyranoside for 5 h at 25°C. .. The fusion protein was purified by affinity chromatography on Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: Paragraph title: 2.1. Protein expression and purification ... The supernatant was applied onto a 50 ml Protein Purification:Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Paragraph title: Protein Purification and in vitro Protein Interaction Assay ... The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: Paragraph title: Protein purification and GST pull-down assay ... Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair Article Snippet: The coding sequences of TDG, Dnmt3a were cloned into pGEX4T-3 (Pharmacia) for expression of the GST-fusions in the E.coli strain BL21 (DE3). .. Protein purification from cells induced with 0.5 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 27°C for 3 h was performed using Positron Emission Tomography:Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Immunoprecipitation:Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B Article Snippet: Paragraph title: GST Pulldown and Immunoprecipitation Assays ... TFIIS-GST was purified by affinity chromatography on Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: Paragraph title: Immunoprecipitation and Western blot analysis ... GST-GULP was pulled down using Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A Article Snippet: Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Fast Protein Liquid Chromatography:Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Lysis:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The lysis was completed by brief ultrasonic pulses using a Sonifier II disrupter (Branson Ultrasonic, Carouge-Geneva, Switzerland). .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Protein extracts were clarified by centrifugation at 13,000 rpm in an Eppendorf microcentrifuge 5415D for 10 min at 4°C, mixed with anti-FLAG M2 agarose (Sigma-Aldrich) and incubated for 2 hr at 4°C. .. The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: When the optical density reached 1.0, they were transferred to the low temperature (16 °C) shaker, and induced with 0.25 mM isopropyl-D-thiogalactoside (IPTG) for 16 h. After that, cells were harvested and then resuspended in lysis buffer (20 mM Tris, pH 7.4, 500 mM NaCl, 10 mM imidazole, 10% glycerol for hexahistidine-tagged fusion protein; 50 mM Tris, pH 7.4, 500 mM NaCl, 2 mM MgCl2 , 5% glycerol for GST-tagged fusion proteins) supplemented with 1 mM PMSF. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation Article Snippet: For the GST pull-down assays equal amounts of fusion proteins were incubated with a defined quantity of cell lysate. .. The proteins bound to the Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: Transfected cells were harvested in 500 μl of NP-40 lysis buffer (25 mM Tris, 137 mM NaCl, 2.7 mM KCl, 1% NP-40, 10% glycerol, 1 mM Na3 VO4 , and Complete Mini EDTA-free Protease Inhibitor Cocktail tablet [Roche, Indianapolis, IN]). .. GST-GULP was pulled down using Article Title: Article Snippet: Briefly, 12 × 106 cells cultured for 6–12 d were lysed by addition of 1.2 ml of lysis buffer (10 mM Tris-HCl, pH 8, 1% Triton X-100, 75 mM NaCl, 5 mM EDTA, and 1 mM dithiothreitol), containing Complete mini protease inhibitors (Roche Diagnostics, Penzberg, Germany) and incubated for 30 min at 4°C. .. After centrifugation at 3500 × g for 10 min at 4°C, the lysate was added to Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch Article Snippet: The whole-cell extract (∼1 mg) was brought up to a volume of 500 μl with lysis buffer containing 2 m m ATP and 25 m m galactose. .. Protein Interaction Assay:Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Paragraph title: Protein Purification and in vitro Protein Interaction Assay ... The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Plasmid Preparation:Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: Immunoblots were visualized by enhanced chemiluminescence method. .. GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: Briefly, the plasmid was transformed into BL21 (DE3) cells and grown in Terrific Broth at 37 °C. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Article Title: Role of the HSP90-Associated Cochaperone p23 in Enhancing Activity of the Androgen Receptor and Significance for Prostate Cancer Article Snippet: Vectors expressing GST fused to full-length p23 (GST-p23); its N terminus (GST-NTD) or empty vector (pGEX-6P-1) was expressed in BL21-codon plus Escherichia coli . .. The GST-fused protein was purified from 5-mg aliquots of supernatant using 200 μl of Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Inserts were sequenced to confirm their fidelity, and then the plasmid constructs were transformed into the Epicurian Coli® BL21-CodonPlus™ strain (Stratagene, Cedar Creek, TX). .. Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae Article Snippet: The resulting plasmid was transformed into Escherichia coli B834(DE3) (Novagen, Madison, Wisconsin, USA). .. The supernatant was applied onto a 50 ml Software:Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Binding Assay:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: Paragraph title: In vitro binding assay ... GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A Article Snippet: Paragraph title: Preparation of recombinant protein, In vitro binding assay and in Vitro kinase reaction ... Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: Paragraph title: ARF6–Sec10 binding assays ... The fusion protein was purified by affinity chromatography on In Vitro:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: PPARγ2-ubiquitin conjugates were prepared by an in vitro ubiquitination assay. .. GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: Paragraph title: In vitro binding assay ... GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity Article Snippet: Paragraph title: Protein Purification and in vitro Protein Interaction Assay ... The agarose beads were collected, washed three times in lysis buffer B (lysis buffer A with 500 mM NaCl), and stored in lysis buffer A. Purification of GST-fused proteins from S. pombe cells was performed as described above except that Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform Article Snippet: Bacteria were subsequently lysed by sonication (model 100 Sonic Dismembrator, Fisher) on ice and cleared by centrifugation at 4 °C for 20 min. Proteins were then purified by incubating cell lysates with a reduced form of Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions Article Snippet: In vitro pull-down experiments : Protein samples were mixed in their correct stoichiometric ratios, using 10 µM She2p wt/ Δhelix E, 10 µM She3p-His6 and 5 µM GST-Myo4-C in a final volume of 100 µl pull-down buffer (20 mM Hepes pH 7.8, 140 mM or 200 mM NaCl, 2 mM MgCl2 , 2 mM DTT). .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Article Title: A Translational Regulator, PUM2, Promotes Both Protein Stability and Kinase Activity of Aurora-A Article Snippet: Paragraph title: Preparation of recombinant protein, In vitro binding assay and in Vitro kinase reaction ... Recombinant His-tagged Aurora-A and GST-tagged PUM2 were induced for 4 hr at room temperature with 1 mM IPTG and purified from the soluble fraction by nickel-agarose (Qiagen) and Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: The fusion protein was purified by affinity chromatography on Size-exclusion Chromatography:Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions Article Snippet: The protein was further purified using ion exchange and size-exclusion chromatography as previously described. .. After centrifugation for 10 min, 16100 × g, 4 °C, 95 µl of the supernatant were incubated with 45 µl Affinity Chromatography:Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B Article Snippet: For IL-6 induction, the cells were serum-starved for at least 16 h, and IL-6 was added at a final concentration of 10 ng/ml for 30 min. .. TFIIS-GST was purified by affinity chromatography on Article Title: Role of α-Globin H Helix in the Building of Tetrameric Human Hemoglobin: Interaction with α-Hemoglobin Stabilizing Protein (AHSP) and Heme Molecule Article Snippet: The obtained solution was incubated in the presence of 1% Triton X-100 for 1 h at 4°C. .. The different supernatants containing soluble GST-AHSPWT /GST-α-Hb complexes were recovered, gazed with CO and purified by a single step of affinity chromatography on Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector Article Snippet: Expression was induced with 0.5 mM isopropyl β-d -thiogalactopyranoside for 5 h at 25°C. .. The fusion protein was purified by affinity chromatography on Article Title: Proteomic analyses and identification of arginine methylated proteins differentially recognized by autosera from anti-Sm positive SLE patients Article Snippet: The CNBP coding sequence was subcloned into pET28b by PCR amplification and Bam HI-Sal I restriction digestion. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Produced:Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions Article Snippet: GST-Nef, sdAb19, and Neffins were produced in Escherichia coli strain BL21 as described previously ( , ). .. Briefly, 1 nmol of recombinant GST or GST-Nef was immobilized on 30 μl of FLAG-tag:Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or Staining:Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation Article Snippet: The proteins bound to the Variant Assay:Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis Article Snippet: For purification of GST-TRF1, GST-Cdk2, His-FLAG-Speedy A, and His-MYC-Cdk2, cDNA encoding mouse TRF1, Cdk2 (variant 1) and Speedy A were cloned into pGEX-4t-1 and modified pET28a (one MYC or FLAG tag was first cloned in to the vector) respectively. .. Then we collected the supernatant by high-speed centrifugation, and incubated them with Ni Sepharose 6 Fast Flow (GE Healthcare, Marlborough, MA) or SDS Page:Article Title: The E3 ubiquitin ligase TRIM23 regulates adipocyte differentiation via stabilization of the adipogenic activator PPARγ Article Snippet: GST pull-down assays were performed according to the instructions of the manufacturer ( Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B Article Snippet: TFIIS-GST was purified by affinity chromatography on Article Title: Peroxiredoxin 6 interferes with TRAIL-induced death-inducing signaling complex formation by binding to death effector domain caspase Article Snippet: GST-fusion proteins cloned into pGEX vector (GE Healthcare, Amersham Biosciences, Uppsala, Sweden) (GST-Prx6, GST-Caspase-10-DED (amino acids 1–219), GST-Caspase-8-DED (1–197), GST-FADD, and GST-CARD (CARD of Apaf-1; 1–601)) were expressed and purified using Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism Article Snippet: GST-GULP was pulled down using Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch Article Snippet: |
