glutathione sepharose 4 fast flow  (GE Healthcare)

 
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    Name:
    Glutathione sepharose 4B
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    Catalog Number:
    GS2376858
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    Structured Review

    GE Healthcare glutathione sepharose 4 fast flow
    Binding of PCSK9 to recombinant EGF-A. A: Purification of GST:EGF-A fusion protein. GST:EGF-A fusion protein was purified using Glutathione <t>Sepharose</t> 4 Fast Flow affinity gel chromatography, followed by size-exclusion chromatography on a Tricorn Superose

    https://www.bioz.com/result/glutathione sepharose 4 fast flow/product/GE Healthcare
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    glutathione sepharose 4 fast flow - by Bioz Stars, 2020-01
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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

    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
    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

    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.
    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:

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    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
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    Article Title: Association of Dnmt3a and thymine DNA glycosylase links DNA methylation with base-excision repair
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    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
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    Article Title: Swi1 Associates with Chromatin through the DDT Domain and Recruits Swi3 to Preserve Genomic Integrity
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    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
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    Article Title: A Ubiquitin-specific Protease Possesses a Decisive Role for Adenovirus Replication and Oncogene-mediated Transformation
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    Article Title: Calcineurin B Homologous Protein 3 Promotes the Biosynthetic Maturation, Cell Surface Stability, and Optimal Transport of the Na+/H+ Exchanger NHE1 Isoform
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    Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions
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    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 glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios. .. Beads were incubated with lysate for 1 h at 4°C, washed three times with lysis buffer, pelleted, and mixed with 100 μl of SDS-sample buffer.

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    Autoradiography:

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    Construct:

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    Ubiquitin Assay:

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    Incubation:

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    Article Title: Transcription Factor IIS Cooperates with the E3 Ligase UBR5 to Ubiquitinate the CDK9 Subunit of the Positive Transcription Elongation Factor B
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    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    Article Title: Dual roles of TRF1 in tethering telomeres to the nuclear envelope and protecting them from fusion during meiosis
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    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 Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    Article Title: Single-Domain Antibody-SH3 Fusions for Efficient Neutralization of HIV-1 Nef Functions
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    Article Title: ASH1 mRNP-core factors form stable complexes in absence of cargo RNA at physiological conditions
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    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 Glutathione-Sepharose beads (Amersham Pharmacia Biotech). .. In in vitro binding assay, recombinant GST-tagged PUM2 (40 µg) on Glutathione-Sepharose beads was incubated with 100 µg purified recombinant His-tagged Aurora-A in binding buffer at 4°C for overnight.

    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 glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios. .. Beads were incubated with lysate for 1 h at 4°C, washed three times with lysis buffer, pelleted, and mixed with 100 μl of SDS-sample buffer.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Expressing:

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    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    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 glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's instruction.

    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 Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    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 glutathione sepharose beads (GE Healthcare).

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    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 glutathione-Sepharose beads (Amersham Biosciences).

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 ). .. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. The lysate was clarified by centrifugation (Beckman JA-17 rotor, 15 krpm, 30 min, 4°C) and mixed with Ni-NTA (Qiagen) beads for 1 hour at 4°C.

    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 glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    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 glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1. .. Beads were washed twice in PBS and then incubated with 1 mg of THP-1 cell lysate for 3 h at 4°C as described previously ( ).

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. .. The beads were pelleted and washed three times with 500 μl of lysis buffer either with or without ATP and galactose.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    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 glutathione-Sepharose beads (Amersham Biosciences).

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 (Glutathione Sepharose 4B, GE Healthcare).

    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 glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    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 glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    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. .. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    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 glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios.

    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 (Glutathione Sepharose 4B, GE Healthcare).

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ). .. GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. 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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    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 Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    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 glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1. .. Beads were washed twice in PBS and then incubated with 1 mg of THP-1 cell lysate for 3 h at 4°C as described previously ( ).

    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 Glutathione-Sepharose beads (Amersham Pharmacia Biotech). .. In in vitro binding assay, recombinant GST-tagged PUM2 (40 µg) on Glutathione-Sepharose beads was incubated with 100 µg purified recombinant His-tagged Aurora-A in binding buffer at 4°C for overnight.

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP.

    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 glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1.

    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 (Glutathione Sepharose 4B, GE Healthcare).

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 glutathione sepharose beads (GE Healthcare).

    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: ... Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry.

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. Expression of GST-CNBP or GST-hnRNP DL fusion proteins in Escherichia coli DH5αor BL21 (DE3) cells was induced with IPTG and purified using Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C. .. The beads were washed, and the protein was eluted using the lysis buffer supplemented with 250 mM imidazole or 10 mM glutathione.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS. .. Proteins that bound glutathione were eluted in 20 m M Tris–HCl pH 7.9, 10 m M reduced glutathione, 1 m M EDTA, 5 m M 2-mercaptoethanol and then dialyzed against 20 m M Tris–HCl pH 7.9, 1 m M EDTA, 5 m M 2-­mercaptoethanol.

    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 glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions. .. Sepharose-bound TFIIS-GST was incubated with HEK 293 whole cell extracts (1 mg) prepared under native conditions ( ).

    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 glutathione-Sepharose 4B (Amersham Biosciences). .. The purified GST-fusion proteins were incubated with 35 S-methionine-labeled proteins, which were translated in vitro using a TNT-coupled transcription/translation system (Promega, Madison, WI, USA) in ice-cold binding buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease inhibitors) at 4°C for 3 h with gentle rocking.

    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The insoluble fraction remaining after solubilization was resuspended in one volume of PBS containing 10% sodium dodecyl sulfate (SDS), sonicated and analyzed by SDS-PAGE and western blotting.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. E. coli BL21(DE3) cells expressing His6 -Swi3 were suspended in lysis buffer H (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 10% Glycerol, 0.25% Tween 20, 10 mM β-mercaptoethanol, and 1 mM PMSF) containing 10 mM imidazole and lysed by sonication using a Branson Digital Sonifier.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's instruction.

    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 Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    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 glutathione sepharose beads (GE Healthcare). .. Equal amounts of beads were incubated with 500 μg of LNCaP or AR-transfected COS-1 cell extract in GST buffer [150 m m NaCl, 20 m m Tris (pH 8), 1 m m EDTA and 0.5% (vol/vol) NP-40] overnight at 4 C before being washed five times in the same buffer before resuspension in sodium dodecyl sulfate loading sample buffer, SDS-PAGE, and immunoblotting.

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    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 Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker.

    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 glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 glutathione Sepharose 4B (Amersham Biosciences) according to the manufacturer's instruction. .. For GST–TDG, the eluate was dialyzed and loaded again onto a Mono S HR 5/5 cation exchange column (Amersham Biosciences) equilibrated with buffer A [25 mM sodium phosphate (pH 7.0), 0.1 M NaCl, 1 mM EDTA, 10% glycerol, 0.01% Triton X-100, 1 mM DTT and 0.25 mM PMSF].

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    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 glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions.

    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 glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    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 Glutathione-Sepharose beads (Amersham Pharmacia Biotech). .. 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 Glutathione-Sepharose beads (Amersham Pharmacia Biotech).

    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The released recombinant proteins were recovered in the supernatant after centrifugation, while GST moiety and Prescission Protease remained bound to the matrix.

    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 (Glutathione Sepharose 4B, GE Healthcare). .. HEK293T cell lysates or PPARγ2-ubiquitin conjugates were incubated with the beads prebound by GST-tagged proteins for 1 hr at 4°C on a rotating platform.

    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) .

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. E. coli BL21(DE3) cells expressing His6 -Swi3 were suspended in lysis buffer H (50 mM NaH2 PO4 pH 8.0, 300 mM NaCl, 10% Glycerol, 0.25% Tween 20, 10 mM β-mercaptoethanol, and 1 mM PMSF) containing 10 mM imidazole and lysed by sonication using a Branson Digital Sonifier.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer. .. The protein samples were then analyzed by SDS-PAGE and Western blotting.

    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 glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ).

    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 glutathione-Sepharose beads (GE Healthcare, München, Germany), previously incubated with 150 μg of GST-fusion proteins or GST at comparable molar ratios.

    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. .. Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. .. The whole-cell extracts were then incubated with 50 μl of 50% glutathione Sepharose beads on a rotator at 4° for 2 hr.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    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 glutathione-Sepharose 4B (Amersham Biosciences). .. The purified GST-fusion proteins were incubated with 35 S-methionine-labeled proteins, which were translated in vitro using a TNT-coupled transcription/translation system (Promega, Madison, WI, USA) in ice-cold binding buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease inhibitors) at 4°C for 3 h with gentle rocking.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 glutathione sepharose beads (GE Healthcare).

    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 glutathione-Sepharose™ beads (Amersham Biosciences) for several hours at 4 °C.

    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 glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose. .. The Ni-NTA beads were washed in lysis buffer H containing 20 mM imidazole, and His6 -Swi3 was eluted with lysis buffer H containing 250 mM imidazole and dialyzed against lysis buffer A. Anti-FLAG M2 agarose beads bound to Swi1-FLAG were mixed with His6 -Swi3 in lysis buffer A, incubated by rotation for 1 hour at 4°C, washed three times in lysis buffer A, and analyzed by Western blotting.

    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 (Glutathione Sepharose 4B, GE Healthcare). .. HEK293T cell lysates or PPARγ2-ubiquitin conjugates were incubated with the beads prebound by GST-tagged proteins for 1 hr at 4°C on a rotating platform.

    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 glutathione-Sepharose 4B (Amersham Biosciences).

    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 Glutathione-Sepharose beads (Amersham Pharmacia Biotech).

    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 glutathione-Sepharose beads (Amersham Biosciences).

    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 (Glutathione Sepharose 4B, GE Healthcare).

    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 glutathione-Sepharose 4B (Amersham Biosciences).

    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 Glutathione Sepharose 4B (GE Healthcare) was used in place of anti-FLAG M2 agarose.

    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. .. The purified GST fusion proteins bound to glutathione-Sepharose beads were washed six times with GST-lysis buffer and then incubated with either 2.5 μl of in vitro translated full-length 35 S-labeled CHP3 or lysates from Chinese hamster ovary (CHO) cells transiently transfected with CHPmyc for several hours at 4 °C.

    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 Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker.

    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 Glutathione-Sepharose beads (Amersham Pharmacia Biotech).

    Article Title: ARF6 controls post-endocytic recycling through its downstream exocyst complex effector
    Article Snippet: The fusion protein was purified by affinity chromatography on glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    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 Glutathione Sepharose beads (GE Healthcare) for 30 min at 4 °C on an overhead shaker.

    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 glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions. .. Sepharose-bound TFIIS-GST was incubated with HEK 293 whole cell extracts (1 mg) prepared under native conditions ( ).

    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 Glutathione Sepharose 4B (GE Healthcare Lifesciences, Uppsala, Sweden) . .. The insoluble fraction remaining after solubilization was resuspended in one volume of PBS containing 10% sodium dodecyl sulfate (SDS), sonicated and analyzed by SDS-PAGE and western blotting.

    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 glutathione-Sepharose beads (Amersham Biosciences). .. After elution with glutathione, the purified protein was dialyzed against 20 mM Tris, pH 7.4, 100 mM NaCl, 2 mM EDTA, 2 mM β-mercaptoethanol, and 10% glycerol, and was stored at −80°C.

    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 Glutathione Sepharose affinity chromatography (GE Amersham Biosciences) according to the manufacturer’s instructions. (His)6 -tagged CNBP protein was prepared from E. coli cells transformed with pET-28b-CNBP. .. The pellet of the extract containing (His)6 -fusion proteins was resuspended with 3 ml Buffer A (6 M Guanidine-HCL, 0.1 M NaH2 PO4 , 0.01 M Tris-HCL, 0.1 M β-mercaptoethanol, 0.01 M PMSF, pH 8.0) at room temperature for 1 hr, followed by centrifugation at 10000 × g for 10 min.

    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 glutathione-Sepharose beads (GE Healthcare) and incubated for 1 h at 4°C with 1, 3, or 9 nmol of recombinant sdAb19, Neffin B6, or Neffin C1.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer. .. The proteins bound to the Glutathione Sepharose (GE Healthcare) were subsequently precipitated by centrifugation (6500 rpm, 5 min, 4°C), six times washed with PBS or lysis buffer, centrifuged and boiled in 25 µl of SDS sample buffer.

    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 Glutathione Sepharose 4B (GE Healthcare, Marlborough, MA) for 2 h at 4 °C.

    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 (Glutathione Sepharose 4B, GE Healthcare). .. The supernatant was collected as an unbound fraction, and nonspecific binding was removed by washing 5 times with ice-cold lysis buffer.

    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 glutathione-Sepharose 4B (GE Healthcare Biosciences) according to the manufacturer's instructions. .. Sepharose-bound TFIIS-GST was incubated with HEK 293 whole cell extracts (1 mg) prepared under native conditions ( ).

    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 glutathione-Sepharose 4B (Amersham Biosciences). .. The purified GST-fusion proteins were incubated with 35 S-methionine-labeled proteins, which were translated in vitro using a TNT-coupled transcription/translation system (Promega, Madison, WI, USA) in ice-cold binding buffer (50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% NP-40, and protease inhibitors) at 4°C for 3 h with gentle rocking.

    Article Title: The adaptor protein GULP promotes Jedi-1–mediated phagocytosis through a clathrin-dependent mechanism
    Article Snippet: GST-GULP was pulled down using glutathione Sepharose beads (Amersham Biosciences, Piscataway, NJ). .. Endogenous Jedi-1 was immunoprecipitated using a previously described polyclonal Jedi-1 antibody ( ).

    Article Title: Genetic Evidence for Sites of Interaction Between the Gal3 and Gal80 Proteins of the Saccharomyces cerevisiae GAL Gene Switch
    Article Snippet: Glutathione Sepharose beads (Amersham Biosciences, Arlington Heights, IL) were equilibrated and resuspended in the lysis buffer as a 50% slurry. .. The beads were pelleted and washed three times with 500 μl of lysis buffer either with or without ATP and galactose.

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    GE Healthcare glutathione sepharose 4 fast flow
    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.
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    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.

    Journal: PLoS Pathogens

    Article Title: Phospholipid scramblase 1 interacts with influenza A virus NP, impairing its nuclear import and thereby suppressing virus replication

    doi: 10.1371/journal.ppat.1006851

    Figure Lengend 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.

    Article Snippet: Then, 300 μl of the cleared lysates from cells transfected with pCAGGS-GST, pCAGGS-GST-PLSCR1, or pCAGGS-GST-NP was mixed with 40 μl of Glutathione Sepharose 4 Fast Flow (GE Healthcare, Pittsburgh, PA) and rocked for 1 h at 4°C.

    Techniques: Co-Immunoprecipitation Assay, Transfection, Expressing, Immunoprecipitation, Western Blot, Pull Down Assay, Construct, Incubation, Flow Cytometry, Staining, RNA Binding Assay, Infection, Magnetic Beads

    Hsp40 interacts with incoming viral ribonucleoprotein complexes in IAV infected cells. ( a ) Hsp40 interacts with NP component of vRNPs. A549 cells were infected with PR8 virus at MOI = 10 in the presence of cycloheximide and cells were harvested 4 h post-infection. Cell extracts were immunoprecipitated using anti-NP, anti-Hsp40, control IgG mouse and rabbit antibodies. RIP was analyzed with quantitative RT-PCR using NP vRNA specific primers. The fold change in NP vRNA pull down levels was calculated with reference to control antibodies IgG mouse and rabbit. ( b ) Cycloheximide (CHX) treatment significantly reduced NP mRNA and vRNA levels in infected cells. The input sample used to set up RNA immunoprecipitation was tested for NP levels in cycloheximide treated and untreated cells by qRT-PCR. The fold change in NP mRNA and vRNA levels was calculated with reference to CHX untreated cells. ( c ) RNase A treatment partially affects the interaction between NP and Hsp40. A549 cells were infected with virus at MOI = 1 and cells were harvested 24 h post-infection. Cell lysate was treated with RNase (100μg/ml) at 4 °C for 45 min followed by immunoprecipitation using anti-Hsp40 antibody. RNA was extracted from remaining cell lysate and was analyzed on formaldehyde agarose gel. ( d ) NP co-localizes with Hsp40 during early stages of infection. A549 cells were mock infected and infected with PR8 virus at MOI = 5 and were fixed at different time intervals. NP and Hsp40 were stained using anti-NP monoclonal antibody and anti-Hsp40 primary antibody, followed by Alexa 594 and Alexa 488 conjugated secondary antibodies respectively. Arrows indicate the co-localization of NP and Hsp40. ( e ) Hsp40 co-localizes with NP and vRNP. A549 cells were mock infected and infected with PR8 virus at MOI = 5 for 4 h and 24 h. NP and Hsp40 were stained using anti-NP antibody and anti-Hsp40 primary antibody followed by Alexa 350 and Alexa 488 conjugated secondary antibodies respectively. NP vRNAs were detected by positive sense RNA probes using FISH. Arrows indicate the co-localization of IAV NP vRNA, NP and Hsp40. Data show mean ± S.D. from one representative experiment (n = 3) of at least three independent experiments.

    Journal: Scientific Reports

    Article Title: Human Heat shock protein 40 (Hsp40/DnaJB1) promotes influenza A virus replication by assisting nuclear import of viral ribonucleoproteins

    doi: 10.1038/srep19063

    Figure Lengend Snippet: Hsp40 interacts with incoming viral ribonucleoprotein complexes in IAV infected cells. ( a ) Hsp40 interacts with NP component of vRNPs. A549 cells were infected with PR8 virus at MOI = 10 in the presence of cycloheximide and cells were harvested 4 h post-infection. Cell extracts were immunoprecipitated using anti-NP, anti-Hsp40, control IgG mouse and rabbit antibodies. RIP was analyzed with quantitative RT-PCR using NP vRNA specific primers. The fold change in NP vRNA pull down levels was calculated with reference to control antibodies IgG mouse and rabbit. ( b ) Cycloheximide (CHX) treatment significantly reduced NP mRNA and vRNA levels in infected cells. The input sample used to set up RNA immunoprecipitation was tested for NP levels in cycloheximide treated and untreated cells by qRT-PCR. The fold change in NP mRNA and vRNA levels was calculated with reference to CHX untreated cells. ( c ) RNase A treatment partially affects the interaction between NP and Hsp40. A549 cells were infected with virus at MOI = 1 and cells were harvested 24 h post-infection. Cell lysate was treated with RNase (100μg/ml) at 4 °C for 45 min followed by immunoprecipitation using anti-Hsp40 antibody. RNA was extracted from remaining cell lysate and was analyzed on formaldehyde agarose gel. ( d ) NP co-localizes with Hsp40 during early stages of infection. A549 cells were mock infected and infected with PR8 virus at MOI = 5 and were fixed at different time intervals. NP and Hsp40 were stained using anti-NP monoclonal antibody and anti-Hsp40 primary antibody, followed by Alexa 594 and Alexa 488 conjugated secondary antibodies respectively. Arrows indicate the co-localization of NP and Hsp40. ( e ) Hsp40 co-localizes with NP and vRNP. A549 cells were mock infected and infected with PR8 virus at MOI = 5 for 4 h and 24 h. NP and Hsp40 were stained using anti-NP antibody and anti-Hsp40 primary antibody followed by Alexa 350 and Alexa 488 conjugated secondary antibodies respectively. NP vRNAs were detected by positive sense RNA probes using FISH. Arrows indicate the co-localization of IAV NP vRNA, NP and Hsp40. Data show mean ± S.D. from one representative experiment (n = 3) of at least three independent experiments.

    Article Snippet: For GST pull down, soluble fraction containing either GST or Hsp40-GST were incubated with glutathione sepharose beads (GST sepharose 4 Fast Flow, GE healthcare).

    Techniques: Infection, Immunoprecipitation, Quantitative RT-PCR, Agarose Gel Electrophoresis, Staining, Fluorescence In Situ Hybridization

    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

    Journal:

    Article Title: Characterization of the role of EGF-A of low density lipoprotein receptor in PCSK9 binding

    doi: 10.1194/jlr.M041129

    Figure Lengend 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

    Article Snippet: The GST:EGF-A fusion protein was purified using Glutathione Sepharose 4 Fast Flow (GE Healthcare) affinity gel chromatography according to the manufacturer's protocol.

    Techniques: Binding Assay, Recombinant, Purification, Flow Cytometry, Chromatography, Size-exclusion Chromatography

    SDS–PAGE analysis. The proteins (0.88 µg) were subjected to SDS–PAGE under reducing conditions. Lane 1, molecular-weight markers (kDa). Lane 2, soluble extract from  E. coli . Lane 3, proteins that bound to glutathione Sepharose 4. Lane 4, protein products after the ‘high-concentration’ trypsin digest. Lane 5, proteins of the major peak eluted from MonoQ. Lane 6, pooled flowthrough fractions from Macro-Prep Ceramic Hydroxyapatite. Lane 7, proteins of the major peak eluted from MonoS. Lane 8, purified PBP 2B transpeptidase after Superdex 75 chromatography.

    Journal: Acta Crystallographica Section F: Structural Biology and Crystallization Communications

    Article Title: Crystallization and preliminary crystallographic analysis of the transpeptidase domain of penicillin-binding protein 2B from Streptococcus pneumoniae

    doi: 10.1107/S1744309108006374

    Figure Lengend Snippet: SDS–PAGE analysis. The proteins (0.88 µg) were subjected to SDS–PAGE under reducing conditions. Lane 1, molecular-weight markers (kDa). Lane 2, soluble extract from E. coli . Lane 3, proteins that bound to glutathione Sepharose 4. Lane 4, protein products after the ‘high-concentration’ trypsin digest. Lane 5, proteins of the major peak eluted from MonoQ. Lane 6, pooled flowthrough fractions from Macro-Prep Ceramic Hydroxyapatite. Lane 7, proteins of the major peak eluted from MonoS. Lane 8, purified PBP 2B transpeptidase after Superdex 75 chromatography.

    Article Snippet: The supernatant was applied onto a 50 ml glutathione Sepharose 4 Fast Flow column (GE Healthcare Biosciences) equilibrated with 1 m M EDTA, 5 m M 2-mercaptoethanol, PBS.

    Techniques: SDS Page, Molecular Weight, Concentration Assay, Purification, Chromatography