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Glutathione Resin, supplied by GE Healthcare, used in various techniques. Bioz Stars score: 99/100, based on 49 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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Clone Assay:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: Protein expression and purification All APOBEC protein encoding sequences were cloned into a pGEX-6P-1 vector (GE Healthcare) with an N-terminal GST tag and PreScission cleavage site. .. After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP).

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: Production of recombinant M2eH-A-S-H in the bacterial expression system Synthetic M2eH-A-S-H ORF was digested with BamHI and EcoRI enzymes and cloned into a commercial vector, pGEX2TK (GE Healthcare). .. The protein was purified by affinity chromatography on glutathione resin (GE Healthcare).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Paragraph title: Cloning, protein expression, and purification ... Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Centrifugation:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: .. After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP). ..

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: .. Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. The supernatant, wash, and SDS eluate (10 μl each) were analyzed by 8% SDS-PAGE and Coomassie Blue staining.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). ..

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). ..

Filtration:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP). .. After centrifugation, the supernatant was analyzed by Superose 6 10/300 GL (GE Healthcare), and further purified by Fast Flow (GE Healthcare) HiLoad 16/60 Superdex 75 gel filtration in large-scale.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Pure fractions were pooled and buffer exchanged by gel filtration on a HiLoad S75 column into 20 mM HEPES pH 7.5, 50 mM NaCl, and 1 mM DTT. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Pure fractions were pooled and buffer exchanged by gel filtration on a HiLoad S75 column into 20 mM HEPES pH 7.5, 50 mM NaCl, and 1 mM DTT. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Construct:

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: A modified pET49 vector was constructed containing EZH2 (residues 421–746) and SUZ12 (residues 535–685), each with an N-terminal GST tag, with the former containing a Precission 3C cleave sequence and the latter thrombin. .. Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Incubation:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: .. After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP). ..

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: .. Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. The supernatant, wash, and SDS eluate (10 μl each) were analyzed by 8% SDS-PAGE and Coomassie Blue staining.

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: Lysates were centrifuged at 12,000 g for 30 min. Glutathione resin (GE Healthcare) was added to the supernatant and incubated for 30 min at 4°C. .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS).

Article Title: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation
Article Snippet: .. Harvested bacteria were lysed (lysis buffer: 25 mM HEPES, 500 mM NaCl, 2 mM DTT, 1% Triton X-100) and incubated with glutathione resin (GE Healthcare). .. After washing extensively, the resin was then incubated with cell lysate from cortical neuronal culture or HEK293T in lysis buffer, obtained by resuspending and sonicating cells in lysis buffer (25 mM HEPES, 150 mM NaCl, 1% Triton X-100, pH 7.4).

Article Title: NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability
Article Snippet: .. Affinity pull-down assays GST-tagged NUCKS1 (3 μg) or GST-tagged RAD51AP1 (5 μg) was incubated with human RAD51 (5 μg) in 30 μl buffer B (25 mM Tris–HCl at pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, 100 mM KCl) on ice for 30 min, and then 15 μl glutathione resin (GE Healthcare) was added. ..

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). .. The GST‐tag was removed by overnight incubation at 4°C with GST‐tagged 3C Precision protease in buffer A. Eluted protein was further purified by anion‐exchange chromatography and gel filtration in buffer A.

Article Title: Discovery and characterization of small molecule Rac1 inhibitors
Article Snippet: .. The supernatant was then incubated with glutathione resin (GE Healthcare) for 2 h at 4°C. ..

Article Title: Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling
Article Snippet: In vitro GST-pull-down assay GST-fusion proteins were produced and purified from bacteria lysates with glutathione resin (GE Healthcare) as described in the Protein Purification section. .. The glutathione beads coupled to GST-fusion proteins were equilibrated with TNET buffer and then incubated with appropriate amount of cell lysates for indicated time at 4 °C.

Article Title: Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation
Article Snippet: GST pull-down assays Various truncations of GST-tagged USP7 proteins were incubated with DNMT1 proteins in binding buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 5% glycerol, 0.05% Triton X-100 and 1 mM dithiothreitol for 2 h at 4 °C. .. The protein samples were then immobilized on 25 μl of glutathione resin (GE Healthcare) for 20 min at 4 °C.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). .. The GST‐tag was removed by overnight incubation at 4°C with GST‐tagged 3C Precision protease in buffer A. Eluted protein was further purified by anion‐exchange chromatography and gel filtration in buffer A.

Activity Assay:

Article Title: Discovery and characterization of small molecule Rac1 inhibitors
Article Snippet: Recombinant PAK1-PBD (70-117aa), WASP-GBD (228-298aa), and Rhotekin-RBD (7-89aa) proteins for Rac1, Cdc42 and RhoA activity assays, respectively, were obtained from Addgene (Cambridge, NH) as glutathione S-transferase (GST) fusion proteins. .. The supernatant was then incubated with glutathione resin (GE Healthcare) for 2 h at 4°C.

Expressing:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: Paragraph title: Protein expression and purification ... After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP).

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: Expression was induced by 0.1 mM IPTG at OD 0.6–0.7. .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS).

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: Paragraph title: Production of recombinant M2eH-A-S-H in the bacterial expression system ... The protein was purified by affinity chromatography on glutathione resin (GE Healthcare).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Paragraph title: Cloning, protein expression, and purification ... Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: Paragraph title: Protein expression and purification ... Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Modification:

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: A modified pET49 vector was constructed containing EZH2 (residues 421–746) and SUZ12 (residues 535–685), each with an N-terminal GST tag, with the former containing a Precission 3C cleave sequence and the latter thrombin. .. Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Western Blot:

Article Title: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation
Article Snippet: Western blotting was performed using rat anti-HA antibody at 1:1,000 (Roche, # 11583816001) For GST-Ubc9 pull-down assay, GST-Ubc9 was expressed in BL21(DE3) E. coli under standard isopropylthiogalactoside induction. .. Harvested bacteria were lysed (lysis buffer: 25 mM HEPES, 500 mM NaCl, 2 mM DTT, 1% Triton X-100) and incubated with glutathione resin (GE Healthcare).

Transformation Assay:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: E. coli cells transformed with the plasmids were grown in LB media at 37 °C until the OD600 reached 0.6. .. After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: USP4 (8–925) wild‐type and C311A mutant were expressed and purified as in . pOPINK‐OTUD1 catalytic domain (residues 287–435) was transformed into Escherichia coli Rosetta 2 pLysS (Novagen) and grown to OD 0.6 followed by induction with 0.2 mM IPTG overnight at 20°C. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: USP4 (8–925) wild‐type and C311A mutant were expressed and purified as in . pOPINK‐OTUD1 catalytic domain (residues 287–435) was transformed into Escherichia coli Rosetta 2 pLysS (Novagen) and grown to OD 0.6 followed by induction with 0.2 mM IPTG overnight at 20°C. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Crystallization Assay:

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: Multiple sequence alignments from different species indicated that EZH2 residues 183–210 (inclusive) were not conserved and this region was removed to aid crystallization. .. Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Flow Cytometry:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP). .. After centrifugation, the supernatant was analyzed by Superose 6 10/300 GL (GE Healthcare), and further purified by Fast Flow (GE Healthcare) HiLoad 16/60 Superdex 75 gel filtration in large-scale.

Chromatography:

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). .. The GST‐tag was removed by overnight incubation at 4°C with GST‐tagged 3C Precision protease in buffer A. Eluted protein was further purified by anion‐exchange chromatography and gel filtration in buffer A.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). .. The GST‐tag was removed by overnight incubation at 4°C with GST‐tagged 3C Precision protease in buffer A. Eluted protein was further purified by anion‐exchange chromatography and gel filtration in buffer A.

Concentration Assay:

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS). .. 20 µl resin saturated with baits was incubated with 1.5 µM prey (MBP-Vps39 or MBP alone; this concentration was chosen to prevent MBP-Vps39 precipitation) in binding buffer (total volume 200 µl) for 30 min at 4°C.

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: The resulting plasmid, pM2eH-A-S-H-GST, was verified by restriction analysis and nucleotide sequencing. pM2eH-A-S-H-GST was used to transform BL21 Escherichia coli strain, and the recombinant strain was used to overproduce M2eH-A-S-H-GST after addition of IPTG (final concentration—1 mM). .. The protein was purified by affinity chromatography on glutathione resin (GE Healthcare).

Sequencing:

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: The resulting plasmid, pM2eH-A-S-H-GST, was verified by restriction analysis and nucleotide sequencing. pM2eH-A-S-H-GST was used to transform BL21 Escherichia coli strain, and the recombinant strain was used to overproduce M2eH-A-S-H-GST after addition of IPTG (final concentration—1 mM). .. The protein was purified by affinity chromatography on glutathione resin (GE Healthcare).

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: A modified pET49 vector was constructed containing EZH2 (residues 421–746) and SUZ12 (residues 535–685), each with an N-terminal GST tag, with the former containing a Precission 3C cleave sequence and the latter thrombin. .. Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Sonication:

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). ..

Article Title: Discovery and characterization of small molecule Rac1 inhibitors
Article Snippet: Lysis was achieved by adding lysozyme (2 mg/mL), 13 mM NaCl, and 1% Triton-X 100 and incubated 50 min at 4°C followed by sonication. .. The supernatant was then incubated with glutathione resin (GE Healthcare) for 2 h at 4°C.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare). ..

Binding Assay:

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. For DNA binding, the single-stranded DNA substrate was 32 P-labeled oligonucleotide P1 (Supplementary Table ).

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS). .. 20 µl resin saturated with baits was incubated with 1.5 µM prey (MBP-Vps39 or MBP alone; this concentration was chosen to prevent MBP-Vps39 precipitation) in binding buffer (total volume 200 µl) for 30 min at 4°C.

Article Title: Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation
Article Snippet: GST pull-down assays Various truncations of GST-tagged USP7 proteins were incubated with DNMT1 proteins in binding buffer containing 25 mM HEPES (pH 7.4), 150 mM NaCl, 5% glycerol, 0.05% Triton X-100 and 1 mM dithiothreitol for 2 h at 4 °C. .. The protein samples were then immobilized on 25 μl of glutathione resin (GE Healthcare) for 20 min at 4 °C.

Ion Exchange Chromatography:

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease. .. Eluted protein was further purified using ion-exchange chromatography (Source Q, GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare) in buffer containing 50 mM Tris pH 8.5, 300 mM NaCl, 1 mM TCEP.

Pull Down Assay:

Article Title: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation
Article Snippet: Western blotting was performed using rat anti-HA antibody at 1:1,000 (Roche, # 11583816001) For GST-Ubc9 pull-down assay, GST-Ubc9 was expressed in BL21(DE3) E. coli under standard isopropylthiogalactoside induction. .. Harvested bacteria were lysed (lysis buffer: 25 mM HEPES, 500 mM NaCl, 2 mM DTT, 1% Triton X-100) and incubated with glutathione resin (GE Healthcare).

Magnetic Beads:

Article Title: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation
Article Snippet: The lysate was subsequently diluted 10-fold with RIPA dilution buffer (25 mM HEPES, 500 mM NaCl, 1% Triton X-100, 0.5% Sodium deoxycholate, pH 7.4), which was used for pull down with 4 μg of anti-SUMO-1 D11 antibody (Santa Cruz, # sc-5308) bound to Protein A/G magnetic beads (Pierce). .. Harvested bacteria were lysed (lysis buffer: 25 mM HEPES, 500 mM NaCl, 2 mM DTT, 1% Triton X-100) and incubated with glutathione resin (GE Healthcare).

Mutagenesis:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP). .. The rA3G-CD1 FWKL and Y124A mutant, hA3G-CD2 and rA3G-CD2 were purified directly by HiLoad 16/60 Superdex 75 gel filtration (GE Healthcare) without PEI treatment.

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C. .. Briefly, equal amounts of glutathione- S -transferase (GST) and fusion proteins consisting of GST fused to truncations of human Caly (NCBI accession no. NP_056537) (Caly-1-94, Caly-104-217, Caly-114-217, Caly-104-155, etc.) or to a double point mutant human Caly (ATEA-104-217) were bound to glutathione resin and blocked with 5% bovine serum albumin in homo­genization buffer.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: USP4 (8–925) wild‐type and C311A mutant were expressed and purified as in . pOPINK‐OTUD1 catalytic domain (residues 287–435) was transformed into Escherichia coli Rosetta 2 pLysS (Novagen) and grown to OD 0.6 followed by induction with 0.2 mM IPTG overnight at 20°C. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: USP4 (8–925) wild‐type and C311A mutant were expressed and purified as in . pOPINK‐OTUD1 catalytic domain (residues 287–435) was transformed into Escherichia coli Rosetta 2 pLysS (Novagen) and grown to OD 0.6 followed by induction with 0.2 mM IPTG overnight at 20°C. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C. .. Briefly, equal amounts of glutathione-S -transferase (GST) and fusion proteins consisting of GST fused to truncations of human Caly (NCBI accession no. NP_056537) (Caly-1-94, Caly-104-217, Caly-114-217, Caly-104-155, etc.) or to a double point mutant human Caly (ATEA-104-217) were bound to glutathione resin and blocked with 5% bovine serum albumin in homo­genization buffer.

Transfection:

Article Title: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation
Article Snippet: For the N2A SUMOylation assay , cells were transfected with HA-SynIa, Flag-Ubc9 and either active YFP-SUMO-1-GG or non-conjugatable YFP-SUMO-ΔGG for 48 h before lysis on ice in buffer containing 20 nM NEM. .. Harvested bacteria were lysed (lysis buffer: 25 mM HEPES, 500 mM NaCl, 2 mM DTT, 1% Triton X-100) and incubated with glutathione resin (GE Healthcare).

Purification:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: Paragraph title: Protein expression and purification ... After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP).

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C. .. Precleared cytosolic fractions of brain (2 mg) or highly purified dynein complex from bovine brain (100 μg) (generous gift of Steve King, University of Central Florida) were added to resins and nutated overnight at 4°C.

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: Recombinant Vps39 was purified with sequential nickel and MBP affinity chromatography according to the manufacturer’s recommendations (resins from GE Healthcare). .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS).

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: .. The protein was purified by affinity chromatography on glutathione resin (GE Healthcare). ..

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Paragraph title: Cloning, protein expression, and purification ... Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Discovery and characterization of small molecule Rac1 inhibitors
Article Snippet: All GST fusion proteins were purified as described previously [ ]. .. The supernatant was then incubated with glutathione resin (GE Healthcare) for 2 h at 4°C.

Article Title: Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling
Article Snippet: .. In vitro GST-pull-down assay GST-fusion proteins were produced and purified from bacteria lysates with glutathione resin (GE Healthcare) as described in the Protein Purification section. .. The glutathione beads coupled to GST-fusion proteins were equilibrated with TNET buffer and then incubated with appropriate amount of cell lysates for indicated time at 4 °C.

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: Paragraph title: Protein expression and purification ... Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C. .. Precleared cytosolic fractions of brain (2 mg) or highly purified dynein complex from bovine brain (100 μg) (generous gift of Steve King, University of Central Florida) were added to resins and nutated overnight at 4°C.

Protein Purification:

Article Title: Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling
Article Snippet: .. In vitro GST-pull-down assay GST-fusion proteins were produced and purified from bacteria lysates with glutathione resin (GE Healthcare) as described in the Protein Purification section. .. The glutathione beads coupled to GST-fusion proteins were equilibrated with TNET buffer and then incubated with appropriate amount of cell lysates for indicated time at 4 °C.

Recombinant:

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: Recombinant Vps39 was purified with sequential nickel and MBP affinity chromatography according to the manufacturer’s recommendations (resins from GE Healthcare). .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS).

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: Paragraph title: Production of recombinant M2eH-A-S-H in the bacterial expression system ... The protein was purified by affinity chromatography on glutathione resin (GE Healthcare).

Article Title: Discovery and characterization of small molecule Rac1 inhibitors
Article Snippet: Paragraph title: Antibodies and recombinant proteins ... The supernatant was then incubated with glutathione resin (GE Healthcare) for 2 h at 4°C.

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: Recombinant PRC2 was much less prone to aggregation when EZH2 was expressed in two parts (EZH2N 1–385 and EZH2C 421–746), removing the linker between the end of MCSS and the beginning of SANT2, thus 28 residues are missing from the middle lobe as indicated in . .. Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Lysis:

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: Beads were washed with lysis buffer (10-column volume) before pull-down assays. .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS).

Article Title: SUMOylation of synapsin Ia maintains synaptic vesicle availability and is reduced in an autism mutation
Article Snippet: .. Harvested bacteria were lysed (lysis buffer: 25 mM HEPES, 500 mM NaCl, 2 mM DTT, 1% Triton X-100) and incubated with glutathione resin (GE Healthcare). .. After washing extensively, the resin was then incubated with cell lysate from cortical neuronal culture or HEK293T in lysis buffer, obtained by resuspending and sonicating cells in lysis buffer (25 mM HEPES, 150 mM NaCl, 1% Triton X-100, pH 7.4).

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Untagged ubiquitin (pET3a) was expressed in Rosetta 2 cells and, after lysis, was treated with 1% v/v perchloric acid to precipitate cellular proteins. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Article Title: Discovery and characterization of small molecule Rac1 inhibitors
Article Snippet: Lysis was achieved by adding lysozyme (2 mg/mL), 13 mM NaCl, and 1% Triton-X 100 and incubated 50 min at 4°C followed by sonication. .. The supernatant was then incubated with glutathione resin (GE Healthcare) for 2 h at 4°C.

Article Title: Active site alanine mutations convert deubiquitinases into high‐affinity ubiquitin‐binding proteins
Article Snippet: Untagged ubiquitin (pET3a) was expressed in Rosetta 2 cells and, after lysis, was treated with 1% v/v perchloric acid to precipitate cellular proteins. .. Cells were lysed by sonication in buffer A (50 mM Tris, 50 mM NaCl, 5 mM DTT, pH 8.5), the lysate was cleared by centrifugation (44,000 × g for 30 min, 4°C) and subjected to a glutathione resin (GE Healthcare).

Mouse Assay:

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: Freshly dissected forebrains from wild-type (WT) c57bl/6 mice were homogenized in eight volumes of homogenization buffer (10 mM HEPES, pH 7.4, 320 mM sucrose) containing protease inhibitors (Thermo, catalogue no. 78430). .. Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C.

Article Title: Dynein Binds and Stimulates Axonal Motility of the Endosome Adaptor and NEEP21 Family Member, Calcyon
Article Snippet: Freshly dissected forebrains from wild-type (WT) c57bl/6 mice were homogenized in eight volumes of homogenization buffer (10mM HEPES, pH 7.4 containing 320 mM sucrose) containing protease inhibitors (Roche). .. Cytosolic fractions were prepared by ultracentrifugation and then pre-cleared with glutathione resin (Amersham Biosciences) added at a 1:10 ratio.

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: GST pull down Freshly dissected forebrains from wild-type (WT) c57bl/6 mice were homogenized in eight volumes of homogenization buffer (10 mM HEPES, pH 7.4, 320 mM sucrose) containing protease inhibitors (Thermo, catalogue no. 78430). .. Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C.

SDS Page:

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. The supernatant, wash, and SDS eluate (10 μl each) were analyzed by 8% SDS-PAGE and Coomassie Blue staining.

Article Title: NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability
Article Snippet: Affinity pull-down assays GST-tagged NUCKS1 (3 μg) or GST-tagged RAD51AP1 (5 μg) was incubated with human RAD51 (5 μg) in 30 μl buffer B (25 mM Tris–HCl at pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, 100 mM KCl) on ice for 30 min, and then 15 μl glutathione resin (GE Healthcare) was added. .. The supernatant, final wash and SDS-eluted fractions (10 μl each) were analyzed by 10% SDS-PAGE and Coomassie Blue staining.

Article Title: Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation
Article Snippet: The protein samples were then immobilized on 25 μl of glutathione resin (GE Healthcare) for 20 min at 4 °C. .. The resin was washed three times with binding buffer, and bound proteins were subjected to SDS–PAGE and stained with Coomassie brilliant blue.

Plasmid Preparation:

Article Title: Crystal structures of APOBEC3G N-domain alone and its complex with DNA
Article Snippet: Protein expression and purification All APOBEC protein encoding sequences were cloned into a pGEX-6P-1 vector (GE Healthcare) with an N-terminal GST tag and PreScission cleavage site. .. After centrifugation, the supernatant of the cell lysates were incubated with glutathione resin (GE Healthcare), and washed with four column volumes of buffer A with 500 mM NaCl, and overnight digestion by PreScission protease in buffer B (500 mM NaCl, 50 mM HEPES pH 7.5, 1 mM TCEP).

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: The resulting plasmid, pM2eH-A-S-H-GST, was verified by restriction analysis and nucleotide sequencing. pM2eH-A-S-H-GST was used to transform BL21 Escherichia coli strain, and the recombinant strain was used to overproduce M2eH-A-S-H-GST after addition of IPTG (final concentration—1 mM). .. The protein was purified by affinity chromatography on glutathione resin (GE Healthcare).

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: A modified pET49 vector was constructed containing EZH2 (residues 421–746) and SUZ12 (residues 535–685), each with an N-terminal GST tag, with the former containing a Precission 3C cleave sequence and the latter thrombin. .. Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease.

Affinity Chromatography:

Article Title: Rab2 promotes autophagic and endocytic lysosomal degradation
Article Snippet: Recombinant Vps39 was purified with sequential nickel and MBP affinity chromatography according to the manufacturer’s recommendations (resins from GE Healthcare). .. For GST pull-down assays, first the glutathione resin (GE Healthcare) with immobilized GST-Rab2 (or GST alone) was equilibrated with binding buffer (20 mM Tris, 50 mM NaCl, 0.1% Triton X-100, 2 mM β-mercaptoethanol, and 200 µM GDP or GTPγS).

Article Title: Presenting Influenza A M2e Antigen on Recombinant Spores of Bacillus subtilis
Article Snippet: .. The protein was purified by affinity chromatography on glutathione resin (GE Healthcare). ..

In Vitro:

Article Title: Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling
Article Snippet: .. In vitro GST-pull-down assay GST-fusion proteins were produced and purified from bacteria lysates with glutathione resin (GE Healthcare) as described in the Protein Purification section. .. The glutathione beads coupled to GST-fusion proteins were equilibrated with TNET buffer and then incubated with appropriate amount of cell lysates for indicated time at 4 °C.

Size-exclusion Chromatography:

Article Title: Structural basis of oncogenic histone H3K27M inhibition of human polycomb repressive complex 2
Article Snippet: Proteins were co-expressed in BL21 (λDE3), immobilized on glutathione resin (GE Healthcare) and separated from tags sequentially, first using alpha thrombin, then with Precission 3C protease. .. Eluted protein was further purified using ion-exchange chromatography (Source Q, GE Healthcare) and size exclusion chromatography (Superdex 200, GE Healthcare) in buffer containing 50 mM Tris pH 8.5, 300 mM NaCl, 1 mM TCEP.

Homogenization:

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: Freshly dissected forebrains from wild-type (WT) c57bl/6 mice were homogenized in eight volumes of homogenization buffer (10 mM HEPES, pH 7.4, 320 mM sucrose) containing protease inhibitors (Thermo, catalogue no. 78430). .. Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C.

Article Title: Dynein Binds and Stimulates Axonal Motility of the Endosome Adaptor and NEEP21 Family Member, Calcyon
Article Snippet: Freshly dissected forebrains from wild-type (WT) c57bl/6 mice were homogenized in eight volumes of homogenization buffer (10mM HEPES, pH 7.4 containing 320 mM sucrose) containing protease inhibitors (Roche). .. Cytosolic fractions were prepared by ultracentrifugation and then pre-cleared with glutathione resin (Amersham Biosciences) added at a 1:10 ratio.

Article Title: Coupling of microtubule motors with AP-3 generated organelles in axons by NEEP21 family member calcyon
Article Snippet: GST pull down Freshly dissected forebrains from wild-type (WT) c57bl/6 mice were homogenized in eight volumes of homogenization buffer (10 mM HEPES, pH 7.4, 320 mM sucrose) containing protease inhibitors (Thermo, catalogue no. 78430). .. Cytosolic fractions were prepared by ultracentrifugation at 100,000 × g for 1 h and then precleared by incubating with glutathione resin (Amersham Biosciences), added at a 1:10 ratio, for 2 h at 4°C.

Produced:

Article Title: Tropomodulin3 is a novel Akt2 effector regulating insulin-stimulated GLUT4 exocytosis through cortical actin remodeling
Article Snippet: .. In vitro GST-pull-down assay GST-fusion proteins were produced and purified from bacteria lysates with glutathione resin (GE Healthcare) as described in the Protein Purification section. .. The glutathione beads coupled to GST-fusion proteins were equilibrated with TNET buffer and then incubated with appropriate amount of cell lysates for indicated time at 4 °C.

Mobility Shift:

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: .. Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. The supernatant, wash, and SDS eluate (10 μl each) were analyzed by 8% SDS-PAGE and Coomassie Blue staining.

Strep-tag:

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. The same procedure was followed in testing UAF1-USP1 and UAF1-FANCI interactions, except that Strep-Tactin resin was used to capture protein complexes via the Strep tag on UAF1 .

Staining:

Article Title: DNA requirement in FANCD2 deubiquitination by USP1-UAF1-RAD51AP1 in the Fanconi anemia DNA damage response
Article Snippet: Affinity pull-down and DNA mobility shift assay To test interaction of UAF1 with RAD51AP1, GST or MBP-tagged RAD51AP1 (2 μg) and Strep II-tagged UAF1 (2 μg) were incubated in 30 µl reaction buffer (25 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl) for 30 min. Then, the reaction was mixed gently with 10 μl glutathione resin (GE Healthcare) or amylose resin (New England Biolabs) at 4 o C for 1 h, which was collected by centrifugation, washed with 50 μl buffer, and treated with 30 μl of 2% SDS to elute bound proteins. .. The supernatant, wash, and SDS eluate (10 μl each) were analyzed by 8% SDS-PAGE and Coomassie Blue staining.

Article Title: NUCKS1 is a novel RAD51AP1 paralog important for homologous recombination and genome stability
Article Snippet: Affinity pull-down assays GST-tagged NUCKS1 (3 μg) or GST-tagged RAD51AP1 (5 μg) was incubated with human RAD51 (5 μg) in 30 μl buffer B (25 mM Tris–HCl at pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, 100 mM KCl) on ice for 30 min, and then 15 μl glutathione resin (GE Healthcare) was added. .. The supernatant, final wash and SDS-eluted fractions (10 μl each) were analyzed by 10% SDS-PAGE and Coomassie Blue staining.

Article Title: Molecular mechanism for USP7-mediated DNMT1 stabilization by acetylation
Article Snippet: The protein samples were then immobilized on 25 μl of glutathione resin (GE Healthcare) for 20 min at 4 °C. .. The resin was washed three times with binding buffer, and bound proteins were subjected to SDS–PAGE and stained with Coomassie brilliant blue.

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    GE Healthcare glutathione sepharose resins
    DDX3 interacts with SHP and HNF4 in vitro and ex vivo . ( a ) Coomassie brilliant blue staining of E. coli BL21 (DE3) expressed recombinant proteins, GST and GST-DDX3. ( b ) In vitro GST pull down assays. Purified GST and GST-DDX3 proteins prebound with <t>glutathione-Sepharose</t> 4B resins were incubated with nuclear extracts (200 μg) of HuH7 and HepG2 cells overexpressing HA-SHP or HA-HNF4 with or without RNase A treatment. Associated proteins were eluted and resolved by SDS-PAGE, then immunoblotted using anti-HA antibody (Roche). Input: nuclear extracts of HuH7 expressed HA-SHP (5%, 10 μg) or HuH7 expressed HA-HNF4 (2.5%, 5 μg). ( c and d ) In vivo co-immunoprecipitation assays. HuH7 cells were cotransfected with plasmids expressing Flag-DDX3 (10 μg) and HA-SHP (10 μg) (panel c) or with Flag-DDX3 (10 μg) and HA-HNF4 (10 μg) (panel d) expression constructs by calcium phosphate co-precipitation method. Nuclear extracts (200 μg) were isolated at 48 hr post-transfection and immunoprecipitated using anti-HA antibody- conjugated agarose beads (panel c) or anti-FLAG M2 affinity resins (panel d). The immunoprecipitates were analyzed by SDS-PAGE, then immunoblotting with anti-FLAG (Sigma) and anti-HA antibodies.
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    DDX3 interacts with SHP and HNF4 in vitro and ex vivo . ( a ) Coomassie brilliant blue staining of E. coli BL21 (DE3) expressed recombinant proteins, GST and GST-DDX3. ( b ) In vitro GST pull down assays. Purified GST and GST-DDX3 proteins prebound with glutathione-Sepharose 4B resins were incubated with nuclear extracts (200 μg) of HuH7 and HepG2 cells overexpressing HA-SHP or HA-HNF4 with or without RNase A treatment. Associated proteins were eluted and resolved by SDS-PAGE, then immunoblotted using anti-HA antibody (Roche). Input: nuclear extracts of HuH7 expressed HA-SHP (5%, 10 μg) or HuH7 expressed HA-HNF4 (2.5%, 5 μg). ( c and d ) In vivo co-immunoprecipitation assays. HuH7 cells were cotransfected with plasmids expressing Flag-DDX3 (10 μg) and HA-SHP (10 μg) (panel c) or with Flag-DDX3 (10 μg) and HA-HNF4 (10 μg) (panel d) expression constructs by calcium phosphate co-precipitation method. Nuclear extracts (200 μg) were isolated at 48 hr post-transfection and immunoprecipitated using anti-HA antibody- conjugated agarose beads (panel c) or anti-FLAG M2 affinity resins (panel d). The immunoprecipitates were analyzed by SDS-PAGE, then immunoblotting with anti-FLAG (Sigma) and anti-HA antibodies.

    Journal: Scientific Reports

    Article Title: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

    doi: 10.1038/srep41452

    Figure Lengend Snippet: DDX3 interacts with SHP and HNF4 in vitro and ex vivo . ( a ) Coomassie brilliant blue staining of E. coli BL21 (DE3) expressed recombinant proteins, GST and GST-DDX3. ( b ) In vitro GST pull down assays. Purified GST and GST-DDX3 proteins prebound with glutathione-Sepharose 4B resins were incubated with nuclear extracts (200 μg) of HuH7 and HepG2 cells overexpressing HA-SHP or HA-HNF4 with or without RNase A treatment. Associated proteins were eluted and resolved by SDS-PAGE, then immunoblotted using anti-HA antibody (Roche). Input: nuclear extracts of HuH7 expressed HA-SHP (5%, 10 μg) or HuH7 expressed HA-HNF4 (2.5%, 5 μg). ( c and d ) In vivo co-immunoprecipitation assays. HuH7 cells were cotransfected with plasmids expressing Flag-DDX3 (10 μg) and HA-SHP (10 μg) (panel c) or with Flag-DDX3 (10 μg) and HA-HNF4 (10 μg) (panel d) expression constructs by calcium phosphate co-precipitation method. Nuclear extracts (200 μg) were isolated at 48 hr post-transfection and immunoprecipitated using anti-HA antibody- conjugated agarose beads (panel c) or anti-FLAG M2 affinity resins (panel d). The immunoprecipitates were analyzed by SDS-PAGE, then immunoblotting with anti-FLAG (Sigma) and anti-HA antibodies.

    Article Snippet: In brief, 20 μl glutathione Sepharose resins (GE Healthcare) prebound GST fusion proteins (3 μg) were incubated with nuclear extracts (200 μg, prepared as described previously ) of HuH7 or HepG2 cells expressing either HA-tagged SHP or HA-tagged HNF4 in the presence or absence of 100 μg/ml RNAse A.

    Techniques: In Vitro, Ex Vivo, Staining, Recombinant, Purification, Incubation, SDS Page, In Vivo, Immunoprecipitation, Expressing, Construct, Isolation, Transfection

    HNF4 interacts with DDX3 through one of its SHP-interacting domains. ( a ) Schematic representation of the GST fusion proteins containing N-terminal, middle and C-terminal regions of HNF4. The binding results of the GST pull down analysis shown in Fig. 6b are summarized. ( b ) Mapping the interaction domains of DDX3 and SHP with HNF4. The GST and GST-HNF4 truncated derivatives prebound with glutathione Sepharose beads were incubated with whole cell lysates (500 μg) from HuH7 cells expressing HA-DDX3 and HA-SHP, respectively. After extensive wash, the bound proteins were resolved by SDS-PAGE and analyzed by Western blot with anti-HA antibody. Lane 5, input: total cell lysate (20 μg) of HuH7 cells expressing HA-tagged DDX3 or HA-tagged SHP. Lane 6, total cell lysate (20 μg) of HuH7 cells. Coomassie brilliant blue staining of the GST-HNF4 truncated derivatives is shown in the lower panel.

    Journal: Scientific Reports

    Article Title: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

    doi: 10.1038/srep41452

    Figure Lengend Snippet: HNF4 interacts with DDX3 through one of its SHP-interacting domains. ( a ) Schematic representation of the GST fusion proteins containing N-terminal, middle and C-terminal regions of HNF4. The binding results of the GST pull down analysis shown in Fig. 6b are summarized. ( b ) Mapping the interaction domains of DDX3 and SHP with HNF4. The GST and GST-HNF4 truncated derivatives prebound with glutathione Sepharose beads were incubated with whole cell lysates (500 μg) from HuH7 cells expressing HA-DDX3 and HA-SHP, respectively. After extensive wash, the bound proteins were resolved by SDS-PAGE and analyzed by Western blot with anti-HA antibody. Lane 5, input: total cell lysate (20 μg) of HuH7 cells expressing HA-tagged DDX3 or HA-tagged SHP. Lane 6, total cell lysate (20 μg) of HuH7 cells. Coomassie brilliant blue staining of the GST-HNF4 truncated derivatives is shown in the lower panel.

    Article Snippet: In brief, 20 μl glutathione Sepharose resins (GE Healthcare) prebound GST fusion proteins (3 μg) were incubated with nuclear extracts (200 μg, prepared as described previously ) of HuH7 or HepG2 cells expressing either HA-tagged SHP or HA-tagged HNF4 in the presence or absence of 100 μg/ml RNAse A.

    Techniques: Binding Assay, Incubation, Expressing, SDS Page, Western Blot, Staining

    DDX3 disrupts the formation of SHP/HNF4 heterodimer and promotes the formation of the active HNF4 homodimer. ( a and b ) HuH7 cells were transfected with HA-SHP expression plasmid alone or together with Flag-HNF4 expression plasmid as indicated. ( a ) Immunoblotting was performed with antibodies against HA and Flag. ( b ) The total cell lysates (2 mg) of co-expressed Flag-HNF4 and HA-SHP were incubated with anti-Flag M2 agarose resins. After extensive washing, increasing amounts of purified GST-DDX3 proteins (as indicated on the top, lane 3–5) were added to the mixtures of the beads with bound fractions. The immunoprecipitated proteins were analyzed by immunoblotting with antibodies against Flag, HA and DDX3. The GST-DDX3 alone (lane 1) and cell lysates of expressed HA-tagged SHP (lane 2) incubated with anti-Flag M2 agarose resins were used as negative control. ( c , d and e ) Similar experiments were performed as shown in panel a and b, except HuH7 cells were transfected with HA-HNF4 expression plasmid alone or together with Flag-SHP expression plasmid ( c and d ), or purified GST protein was used instead of GST-DDX3 in ( e ). ( f ) Overexpression of DDX3 increases HNF4 homodimer formation. HuH7 cells were transfected with either GFP or GFP-DDX3 expressing plasmids together with HA and Flag vectors (−) or expressing plasmids for HA-HNF4 and Flag-HNF4 (+). Forty-eight hours later, cells were fixed and analyzed by PLA with anti-HA (Abcam) and anti-Flag antibodies. Nuclei were stained with DAPI (blue). In cells cotransfected with HA-HNF4 and Flag-HNF4, the numbers of PLA signals per cell were quantified using MetaMorph software and are shown as means ± S.D. relative to that of GFP-cotransfected cells. n = 50. *** p

    Journal: Scientific Reports

    Article Title: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

    doi: 10.1038/srep41452

    Figure Lengend Snippet: DDX3 disrupts the formation of SHP/HNF4 heterodimer and promotes the formation of the active HNF4 homodimer. ( a and b ) HuH7 cells were transfected with HA-SHP expression plasmid alone or together with Flag-HNF4 expression plasmid as indicated. ( a ) Immunoblotting was performed with antibodies against HA and Flag. ( b ) The total cell lysates (2 mg) of co-expressed Flag-HNF4 and HA-SHP were incubated with anti-Flag M2 agarose resins. After extensive washing, increasing amounts of purified GST-DDX3 proteins (as indicated on the top, lane 3–5) were added to the mixtures of the beads with bound fractions. The immunoprecipitated proteins were analyzed by immunoblotting with antibodies against Flag, HA and DDX3. The GST-DDX3 alone (lane 1) and cell lysates of expressed HA-tagged SHP (lane 2) incubated with anti-Flag M2 agarose resins were used as negative control. ( c , d and e ) Similar experiments were performed as shown in panel a and b, except HuH7 cells were transfected with HA-HNF4 expression plasmid alone or together with Flag-SHP expression plasmid ( c and d ), or purified GST protein was used instead of GST-DDX3 in ( e ). ( f ) Overexpression of DDX3 increases HNF4 homodimer formation. HuH7 cells were transfected with either GFP or GFP-DDX3 expressing plasmids together with HA and Flag vectors (−) or expressing plasmids for HA-HNF4 and Flag-HNF4 (+). Forty-eight hours later, cells were fixed and analyzed by PLA with anti-HA (Abcam) and anti-Flag antibodies. Nuclei were stained with DAPI (blue). In cells cotransfected with HA-HNF4 and Flag-HNF4, the numbers of PLA signals per cell were quantified using MetaMorph software and are shown as means ± S.D. relative to that of GFP-cotransfected cells. n = 50. *** p

    Article Snippet: In brief, 20 μl glutathione Sepharose resins (GE Healthcare) prebound GST fusion proteins (3 μg) were incubated with nuclear extracts (200 μg, prepared as described previously ) of HuH7 or HepG2 cells expressing either HA-tagged SHP or HA-tagged HNF4 in the presence or absence of 100 μg/ml RNAse A.

    Techniques: Transfection, Expressing, Plasmid Preparation, Incubation, Purification, Immunoprecipitation, Negative Control, Over Expression, Proximity Ligation Assay, Staining, Software

    DDX3 interacts with CBP/p300 and induces the acetylation status of HNF4. ( a ) DDX3 interacts with CBP and p300 in vivo . HuH7 cells were transfected with HA-DDX3 expressing plasmid or vector control. After 48 hr, the nuclear fractions (200 μg) were collected and incubated with antibodies against CBP or p300, and then the mixtures were bound to protein G sepharose beads. The precipitates were subjected to immunoblotting with anti-HA antibody. The IgG-conjugated protein G sepharose beads (mouse, lane 3 and lane 8) incubated with HA-DDX3 expressed nuclear extracts were used as negative control. Input; 10% (20 μg) of the nuclear extract. ( b ) Ectopic expression of DDX3 induces the acetylation status of HA-HNF4. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. The whole cell extracts (1 mg) prepared from the transfected cells were subjected to immunoprecipitation of HA-HNF4 with anti-HA agarose beads. After extensive wash, the total amounts and acetylated forms of immunoprecipitated HA-HNF4 were detected by Western blot analysis with anti-HA and anti-acetylated lysine (Cell Signaling Technology) antibodies, respectively. ( c ) In situ proximity ligation assay (PLA) indicates that ectopic expression of DDX3 induces endogenous HNF4 acetylation. HuH7 cells transfected with either GFP or GFP-DDX3 expressing plasmids for 48 hr were fixed and subjected to PLA with anti-HNF4 (Abcam) and anti-acetylated lysine antibodies. Anti-NS5A antibody (Austral Biologicals) served as control. Nuclei were stained with DAPI (blue). The numbers of PLA signals per cell were quantified using MetaMorph software (Molecular Devices) and are shown as means ± S.D. relative to that of GFP-transfected cells. n = 50. *** p

    Journal: Scientific Reports

    Article Title: RNA helicase DDX3 maintains lipid homeostasis through upregulation of the microsomal triglyceride transfer protein by interacting with HNF4 and SHP

    doi: 10.1038/srep41452

    Figure Lengend Snippet: DDX3 interacts with CBP/p300 and induces the acetylation status of HNF4. ( a ) DDX3 interacts with CBP and p300 in vivo . HuH7 cells were transfected with HA-DDX3 expressing plasmid or vector control. After 48 hr, the nuclear fractions (200 μg) were collected and incubated with antibodies against CBP or p300, and then the mixtures were bound to protein G sepharose beads. The precipitates were subjected to immunoblotting with anti-HA antibody. The IgG-conjugated protein G sepharose beads (mouse, lane 3 and lane 8) incubated with HA-DDX3 expressed nuclear extracts were used as negative control. Input; 10% (20 μg) of the nuclear extract. ( b ) Ectopic expression of DDX3 induces the acetylation status of HA-HNF4. HuH7 cells were transfected with plasmid expressing HA-HNF4 alone or together with HA-DDX3 expression construct as indicated. The whole cell extracts (1 mg) prepared from the transfected cells were subjected to immunoprecipitation of HA-HNF4 with anti-HA agarose beads. After extensive wash, the total amounts and acetylated forms of immunoprecipitated HA-HNF4 were detected by Western blot analysis with anti-HA and anti-acetylated lysine (Cell Signaling Technology) antibodies, respectively. ( c ) In situ proximity ligation assay (PLA) indicates that ectopic expression of DDX3 induces endogenous HNF4 acetylation. HuH7 cells transfected with either GFP or GFP-DDX3 expressing plasmids for 48 hr were fixed and subjected to PLA with anti-HNF4 (Abcam) and anti-acetylated lysine antibodies. Anti-NS5A antibody (Austral Biologicals) served as control. Nuclei were stained with DAPI (blue). The numbers of PLA signals per cell were quantified using MetaMorph software (Molecular Devices) and are shown as means ± S.D. relative to that of GFP-transfected cells. n = 50. *** p

    Article Snippet: In brief, 20 μl glutathione Sepharose resins (GE Healthcare) prebound GST fusion proteins (3 μg) were incubated with nuclear extracts (200 μg, prepared as described previously ) of HuH7 or HepG2 cells expressing either HA-tagged SHP or HA-tagged HNF4 in the presence or absence of 100 μg/ml RNAse A.

    Techniques: In Vivo, Transfection, Expressing, Plasmid Preparation, Incubation, Negative Control, Construct, Immunoprecipitation, Western Blot, In Situ, Proximity Ligation Assay, Staining, Software

    Co-immunoprecipitation of U3 snoRNA with ProtA-Nop58 in Δ nop17 cells. (A) Total cell extracts from strains WT, Δ nop17 and Δ rsa1 were mixed with IgG-Sepharose beads for co-immunoprecipitation of snoRNAs with ProtA-Nop58. Bound RNA was detected by northern blotting using probes specific to the snoRNA U3. Membrane was washed and re-hybridized against probe specific to the 5S rRNA (internal control). TE, total extract; B, bound fraction. Bands were quantitated using Typhoon equipment and mean values of three biological replicates are shown. U3 bands were quantitated relative to 5S bands in each lane. U3/5S in mutants were then calculated relative to WT strain. (B) Nop58 immunoprecipitates snoRNA U3 chromatin. ChIP assay with A-Nop58 expressed in strains WT, Δ nop17 and Δ rsa1 was performed, followed by RT-qPCR reactions with primers for amplification of various regions of the U3 snoRNA gene. Mean values are based on three different experiments with two biological replicates.

    Journal: BMC Molecular Biology

    Article Title: Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex

    doi: 10.1186/s12867-015-0037-5

    Figure Lengend Snippet: Co-immunoprecipitation of U3 snoRNA with ProtA-Nop58 in Δ nop17 cells. (A) Total cell extracts from strains WT, Δ nop17 and Δ rsa1 were mixed with IgG-Sepharose beads for co-immunoprecipitation of snoRNAs with ProtA-Nop58. Bound RNA was detected by northern blotting using probes specific to the snoRNA U3. Membrane was washed and re-hybridized against probe specific to the 5S rRNA (internal control). TE, total extract; B, bound fraction. Bands were quantitated using Typhoon equipment and mean values of three biological replicates are shown. U3 bands were quantitated relative to 5S bands in each lane. U3/5S in mutants were then calculated relative to WT strain. (B) Nop58 immunoprecipitates snoRNA U3 chromatin. ChIP assay with A-Nop58 expressed in strains WT, Δ nop17 and Δ rsa1 was performed, followed by RT-qPCR reactions with primers for amplification of various regions of the U3 snoRNA gene. Mean values are based on three different experiments with two biological replicates.

    Article Snippet: For the Snu13-Nop1-Nop17 pull-down, the total extracts containing GST or GST-Snu13 were incubated for 1 hour at 4°C with glutathione-sepharose resin (GE Healthcare) in PBS buffer.

    Techniques: Immunoprecipitation, Northern Blot, Chromatin Immunoprecipitation, Quantitative RT-PCR, Amplification

    Nop58 interacts with Nop17 through its C-terminal portion. (A) Schematics summarizing the results of two-hybrid assay with deletion mutants of Nop58 and Nop17. Nop58(324–512) mutant interacts with Nop17. (B) Co-immunoprecipitation of RNA with ProtA-Nop58 in the absence or presence of different amounts of Nop17. Incubation of total extract with IgG-sepharose beads was performed for 2 h at 4°C in the presence or absence of purified GST-Nop17. Co-immunoprecipitated U3 snoRNA was detected by northern blot. 5S rRNA was used as an internal control. (C) Quantitation of the U3 bands corrected by 5S bands after northern hybridization.

    Journal: BMC Molecular Biology

    Article Title: Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex

    doi: 10.1186/s12867-015-0037-5

    Figure Lengend Snippet: Nop58 interacts with Nop17 through its C-terminal portion. (A) Schematics summarizing the results of two-hybrid assay with deletion mutants of Nop58 and Nop17. Nop58(324–512) mutant interacts with Nop17. (B) Co-immunoprecipitation of RNA with ProtA-Nop58 in the absence or presence of different amounts of Nop17. Incubation of total extract with IgG-sepharose beads was performed for 2 h at 4°C in the presence or absence of purified GST-Nop17. Co-immunoprecipitated U3 snoRNA was detected by northern blot. 5S rRNA was used as an internal control. (C) Quantitation of the U3 bands corrected by 5S bands after northern hybridization.

    Article Snippet: For the Snu13-Nop1-Nop17 pull-down, the total extracts containing GST or GST-Snu13 were incubated for 1 hour at 4°C with glutathione-sepharose resin (GE Healthcare) in PBS buffer.

    Techniques: Two Hybrid Assay, Mutagenesis, Immunoprecipitation, Incubation, Purification, Northern Blot, Quantitation Assay, Hybridization

    Interaction of Nop17 with the other subunits of the R 2 TP complex. (A) Analysis of the interactions between Nop17 and Rvb1, and Rvb2 through the two-hybrid assay. BD-Nop17 interacts with both AD-Rvb1 and AD-Rvb2, as seen by the expression of the reporter genes HIS3 and lacZ . BD-Rvb2 + AD-Nop17 is stronger than BD-Rvb1-AD-Nop17. BD-Nip7/AD-Rrp43 and BD-Nip7/AD-Nop8 were used as positive controls for interaction [ 21 , 22 ] (B) Pull-down assay to confirm direct interaction between Nop17 and Rvb1/2. GST or GST-Nop17 were bound to glutathione-sepharose beads, followed by the incubation with His-Rvb1, or His-Rvb2, in the absence, or presence of 1 mM ATP or ADP at 4°C for 2 hours. Fractions from total extract (TE), flow through (FT), wash (W), or bound (B) were separated by SDS-PAGE and subjected to western blot with anti-His or anti-GST sera. Interaction Nop17-Rvb2 is independent of ATP. (C) Two-hybrid assay for the analysis of Nop17-Tah1 and Nop17-Hsp90 interaction. BD-Nop17 did not interact with AD-Hsp90, whereas BD-Nop17 interacted with AD-Tah1. Mutation of Nop17 in the position 306 disrupts interaction with Tah1.

    Journal: BMC Molecular Biology

    Article Title: Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex

    doi: 10.1186/s12867-015-0037-5

    Figure Lengend Snippet: Interaction of Nop17 with the other subunits of the R 2 TP complex. (A) Analysis of the interactions between Nop17 and Rvb1, and Rvb2 through the two-hybrid assay. BD-Nop17 interacts with both AD-Rvb1 and AD-Rvb2, as seen by the expression of the reporter genes HIS3 and lacZ . BD-Rvb2 + AD-Nop17 is stronger than BD-Rvb1-AD-Nop17. BD-Nip7/AD-Rrp43 and BD-Nip7/AD-Nop8 were used as positive controls for interaction [ 21 , 22 ] (B) Pull-down assay to confirm direct interaction between Nop17 and Rvb1/2. GST or GST-Nop17 were bound to glutathione-sepharose beads, followed by the incubation with His-Rvb1, or His-Rvb2, in the absence, or presence of 1 mM ATP or ADP at 4°C for 2 hours. Fractions from total extract (TE), flow through (FT), wash (W), or bound (B) were separated by SDS-PAGE and subjected to western blot with anti-His or anti-GST sera. Interaction Nop17-Rvb2 is independent of ATP. (C) Two-hybrid assay for the analysis of Nop17-Tah1 and Nop17-Hsp90 interaction. BD-Nop17 did not interact with AD-Hsp90, whereas BD-Nop17 interacted with AD-Tah1. Mutation of Nop17 in the position 306 disrupts interaction with Tah1.

    Article Snippet: For the Snu13-Nop1-Nop17 pull-down, the total extracts containing GST or GST-Snu13 were incubated for 1 hour at 4°C with glutathione-sepharose resin (GE Healthcare) in PBS buffer.

    Techniques: Two Hybrid Assay, Expressing, Pull Down Assay, Incubation, Flow Cytometry, SDS Page, Western Blot, Mutagenesis

    Interaction between Nop17 and other C/D box snoRNP subunits. (A) Protein pull-down to visualize the interaction between Nop1 and Snu13. Total extracts from E. coli cells expressing GST or GST-Snu13 (input) were first incubated with GST-sepharose beads. Extracts from cells expressing His-Nop1 (input) were then added to the beads. Flow through was collected (FT) after the addition of each extract, and beads were washed. Proteins were eluted with reduced glutathione (Elu). Samples from each fraction were subjected to SDS-PAGE and western blot with anti-GST and anti-His sera. Elution fractions are indicated by arrows. (B) Nop17 interacts with the complex Nop1/Snu13 in pull-down assays. His-Nop1 was added to either GST or GST-Snu13 immobilized in glutathione-sepharose beads. After that, His-Nop17 was added to the beads, and was pulled-down only by the GST-Snu13/His-Nop1 complex. Bands were visualized by western blot with anti-GST and anti-His sera.

    Journal: BMC Molecular Biology

    Article Title: Nop17 is a key R2TP factor for the assembly and maturation of box C/D snoRNP complex

    doi: 10.1186/s12867-015-0037-5

    Figure Lengend Snippet: Interaction between Nop17 and other C/D box snoRNP subunits. (A) Protein pull-down to visualize the interaction between Nop1 and Snu13. Total extracts from E. coli cells expressing GST or GST-Snu13 (input) were first incubated with GST-sepharose beads. Extracts from cells expressing His-Nop1 (input) were then added to the beads. Flow through was collected (FT) after the addition of each extract, and beads were washed. Proteins were eluted with reduced glutathione (Elu). Samples from each fraction were subjected to SDS-PAGE and western blot with anti-GST and anti-His sera. Elution fractions are indicated by arrows. (B) Nop17 interacts with the complex Nop1/Snu13 in pull-down assays. His-Nop1 was added to either GST or GST-Snu13 immobilized in glutathione-sepharose beads. After that, His-Nop17 was added to the beads, and was pulled-down only by the GST-Snu13/His-Nop1 complex. Bands were visualized by western blot with anti-GST and anti-His sera.

    Article Snippet: For the Snu13-Nop1-Nop17 pull-down, the total extracts containing GST or GST-Snu13 were incubated for 1 hour at 4°C with glutathione-sepharose resin (GE Healthcare) in PBS buffer.

    Techniques: Expressing, Incubation, Flow Cytometry, SDS Page, Western Blot

    The MEK2-E2 interaction occurs during the late stage of CSFV replication and requires the C termini of MEK2 and E2. (A) Co-IP analysis of E2 and MEK2 in CSFV-infected cells. PK-15 cells were infected with CSFV, and the cells lysates were collected at 24, 48, and 60 h postinfection (hpi) and precleared with protein G-agarose, followed by incubation with a rabbit anti-phosphorylated MEK2 (anti-p-MEK2) or a rabbit anti-MEK2 monoclonal antibody (MAb) (1:500) at 4°C for 6 h and incubation with protein G-agarose at 4°C for 6 h. The bound proteins in the agarose were examined by Western blotting using anti-E2 MAb WH303 (1:200). (B) Schematic representation of the porcine MEK2 protein domains and individual MEK2 deletion mutants. (C) GST pulldown analysis of the interaction of GST-tagged MEK2 or its mutants with Flag-tagged E2 expressed in HEK293T cells. The recombinant protein of GST-MEK2 or GST-tagged MEK2 mutants was incubated with glutathione-Sepharose 4B resin (catalog no. 17-0756-01; GE Healthcare). The resin was washed with PBS and incubated with the Flag-tagged E2 protein expressed in HEK293T cells. Immunoblotting analysis was conducted to detect the bound proteins. (D) Schematic representation of the truncated E2 mutants. (E) GST pulldown analysis of GST-tagged MEK2 and Flag-tagged, truncated E2 mutants. The recombinant protein GST-MEK2 was incubated with glutathione-Sepharose 4B resin and then with a series of Flag-tagged, truncated E2 mutants. The bound proteins in the resin were then analyzed by Western blotting as described above.

    Journal: Journal of Virology

    Article Title: Mitogen-Activated Protein Kinase Kinase 2, a Novel E2-Interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway

    doi: 10.1128/JVI.01407-16

    Figure Lengend Snippet: The MEK2-E2 interaction occurs during the late stage of CSFV replication and requires the C termini of MEK2 and E2. (A) Co-IP analysis of E2 and MEK2 in CSFV-infected cells. PK-15 cells were infected with CSFV, and the cells lysates were collected at 24, 48, and 60 h postinfection (hpi) and precleared with protein G-agarose, followed by incubation with a rabbit anti-phosphorylated MEK2 (anti-p-MEK2) or a rabbit anti-MEK2 monoclonal antibody (MAb) (1:500) at 4°C for 6 h and incubation with protein G-agarose at 4°C for 6 h. The bound proteins in the agarose were examined by Western blotting using anti-E2 MAb WH303 (1:200). (B) Schematic representation of the porcine MEK2 protein domains and individual MEK2 deletion mutants. (C) GST pulldown analysis of the interaction of GST-tagged MEK2 or its mutants with Flag-tagged E2 expressed in HEK293T cells. The recombinant protein of GST-MEK2 or GST-tagged MEK2 mutants was incubated with glutathione-Sepharose 4B resin (catalog no. 17-0756-01; GE Healthcare). The resin was washed with PBS and incubated with the Flag-tagged E2 protein expressed in HEK293T cells. Immunoblotting analysis was conducted to detect the bound proteins. (D) Schematic representation of the truncated E2 mutants. (E) GST pulldown analysis of GST-tagged MEK2 and Flag-tagged, truncated E2 mutants. The recombinant protein GST-MEK2 was incubated with glutathione-Sepharose 4B resin and then with a series of Flag-tagged, truncated E2 mutants. The bound proteins in the resin were then analyzed by Western blotting as described above.

    Article Snippet: GST-tagged MEK2 mutants were expressed in Escherichia coli BL21(DE3) cells and incubated with glutathione-Sepharose 4B resin (catalog no. 17-0756-01; GE Healthcare).

    Techniques: Co-Immunoprecipitation Assay, Infection, Incubation, Western Blot, Recombinant

    Interaction between the CSFV E2 protein and MEK2. (A) Yeast cotransformation assay. The Y2HGold yeast strain was cotransformed with pGBKT7-E2 (BD-E2)/pGADT7-MEK2 (AD-MEK2), pGBKT7-p53 (BD-p53)/pGADT7-T (AD-T) (positive control), or pGBKT7-Lamin (BD-Lamin)/AD-T (negative control). (B) Coimmunoprecipitation (Co-IP) analysis of MEK2 and E2. HEK293T cells were cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag. Cells collected at 48 h posttransfection (hpt) were lysed, precleared with protein G-agarose, and incubated with anti-Flag M2 affinity gel for 6 h at 4°C. The proteins were analyzed by Western blotting using a rabbit anti-Flag or anti-Myc polyclonal antibody (1:500). (C) GST pulldown assay. GST or GST-E2 expressed in Escherichia coli BL21(DE3) was purified with a glutathione-Sepharose 4B resin (catalog no. 10049253; GE Healthcare) and incubated with Myc-MEK2 expressed in HEK293T cells. The bound proteins were subjected to Western blotting using the indicated antibodies. (D and E) Colocalization of MEK2 with E2. HEK293T cells cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag (D) or PK-15 cells infected with CSFV (E) were examined by indirect immunofluorescence assay (IFA) for expression of MEK2 (red) and E2 (green).

    Journal: Journal of Virology

    Article Title: Mitogen-Activated Protein Kinase Kinase 2, a Novel E2-Interacting Protein, Promotes the Growth of Classical Swine Fever Virus via Attenuation of the JAK-STAT Signaling Pathway

    doi: 10.1128/JVI.01407-16

    Figure Lengend Snippet: Interaction between the CSFV E2 protein and MEK2. (A) Yeast cotransformation assay. The Y2HGold yeast strain was cotransformed with pGBKT7-E2 (BD-E2)/pGADT7-MEK2 (AD-MEK2), pGBKT7-p53 (BD-p53)/pGADT7-T (AD-T) (positive control), or pGBKT7-Lamin (BD-Lamin)/AD-T (negative control). (B) Coimmunoprecipitation (Co-IP) analysis of MEK2 and E2. HEK293T cells were cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag. Cells collected at 48 h posttransfection (hpt) were lysed, precleared with protein G-agarose, and incubated with anti-Flag M2 affinity gel for 6 h at 4°C. The proteins were analyzed by Western blotting using a rabbit anti-Flag or anti-Myc polyclonal antibody (1:500). (C) GST pulldown assay. GST or GST-E2 expressed in Escherichia coli BL21(DE3) was purified with a glutathione-Sepharose 4B resin (catalog no. 10049253; GE Healthcare) and incubated with Myc-MEK2 expressed in HEK293T cells. The bound proteins were subjected to Western blotting using the indicated antibodies. (D and E) Colocalization of MEK2 with E2. HEK293T cells cotransfected with pMyc-MEK2 and pCAGGS-E2-Flag (D) or PK-15 cells infected with CSFV (E) were examined by indirect immunofluorescence assay (IFA) for expression of MEK2 (red) and E2 (green).

    Article Snippet: GST-tagged MEK2 mutants were expressed in Escherichia coli BL21(DE3) cells and incubated with glutathione-Sepharose 4B resin (catalog no. 17-0756-01; GE Healthcare).

    Techniques: Positive Control, Negative Control, Co-Immunoprecipitation Assay, Incubation, Western Blot, GST Pulldown Assay, Purification, Infection, Immunofluorescence, Expressing

    Bivalent scFv-Fc antibodies are internalized into cells in the FGFR1-dependent manner. ( a ) Antibodies in the bivalent scFv-Fc format are internalized into cells that overproduce FGFR1. Serum starved U2OSR1 cells, that overproduce FGFR1 were incubated with FGF1.myc, scFv.myc proteins or scFv-Fc antibody fragments to allow for formation of antibody-FGFR1 and FGF1.myc-FGFR1 complexes. Cells were then shifted for 45 min to 37 °C to initiate internalization. The internalization reaction was stopped by cooling down the cells on ice. The surface bound FGF1.myc or antibody fragments were removed by washing with low pH buffer containing high salt concentration (HSLP) and internalized FGF1.myc, scFv.myc and scFv-Fc proteins were recovered from cell lysates with anti-c-Myc (for FGF1.myc and scFv proteins) and Protein A Sepharose (for scFv-Fc proteins). Internalized proteins were detected by Western blotting using specific antibodies. ( b ) Internalization of scFv-Fc antibody fragments is strictly dependent on the level of FGFR1. Control U2OS cells that contain only very low level of FGFR1 were not able to internalize scFv-Fc proteins. ( c ) FGF1 and bivalent scFv-Fc antibody fragments stimulate FGFR1 degradation. Serum starved U2OSR1 cells were incubated with FGF1, scFvD2 or scFvD2-Fc in the presence or absence of cycloheximide (CHX). Cells were lysed and the level of FGFR1 was assessed by Western blotting. Tubulin was used as a loading control. Cropped blots were displayed, full size blots are included in Supplementary Information.

    Journal: Scientific Reports

    Article Title: Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity

    doi: 10.1038/s41598-017-07479-z

    Figure Lengend Snippet: Bivalent scFv-Fc antibodies are internalized into cells in the FGFR1-dependent manner. ( a ) Antibodies in the bivalent scFv-Fc format are internalized into cells that overproduce FGFR1. Serum starved U2OSR1 cells, that overproduce FGFR1 were incubated with FGF1.myc, scFv.myc proteins or scFv-Fc antibody fragments to allow for formation of antibody-FGFR1 and FGF1.myc-FGFR1 complexes. Cells were then shifted for 45 min to 37 °C to initiate internalization. The internalization reaction was stopped by cooling down the cells on ice. The surface bound FGF1.myc or antibody fragments were removed by washing with low pH buffer containing high salt concentration (HSLP) and internalized FGF1.myc, scFv.myc and scFv-Fc proteins were recovered from cell lysates with anti-c-Myc (for FGF1.myc and scFv proteins) and Protein A Sepharose (for scFv-Fc proteins). Internalized proteins were detected by Western blotting using specific antibodies. ( b ) Internalization of scFv-Fc antibody fragments is strictly dependent on the level of FGFR1. Control U2OS cells that contain only very low level of FGFR1 were not able to internalize scFv-Fc proteins. ( c ) FGF1 and bivalent scFv-Fc antibody fragments stimulate FGFR1 degradation. Serum starved U2OSR1 cells were incubated with FGF1, scFvD2 or scFvD2-Fc in the presence or absence of cycloheximide (CHX). Cells were lysed and the level of FGFR1 was assessed by Western blotting. Tubulin was used as a loading control. Cropped blots were displayed, full size blots are included in Supplementary Information.

    Article Snippet: Protein A Sepharose and Glutathione Sepharose resins were from GE Healthcare (Piscataway, NJ, USA).

    Techniques: Incubation, Concentration Assay, Western Blot

    Antibody fragments bind to the domain D1 of FGFR1. ( a ) Schematic representation of anti-FGFR1 antibody fragments used in this study. The scFv antibody format contains single antigen binding site formed by variable domains of heavy and light antibody chains (VH and VL), whereas scFv-Fc antibody fragments contain two identical binding sites for antigen fused by constant domains of heavy chain of human IgG1 (CH2 and CH3). ( b ) Analyzed antibodies recognize epitopes within domain D1 of FGFR1. scFvD2.myc was bound to the anti-c-Myc agarose and incubated with either purified full length extracellular part of FGFR1 fused with Fc fragment (FGFR1 D1-D2-D3-Fc) or with the Fc-fusion of the extracellular part of FGFR1 lacking domain D1 (FGFR1 D2-D3-Fc). Proteins bound to scFvD2.myc were analyzed with anti-Fc antibodies. ( c ) Direct Interaction of scFv’s with the domain D1 of the FGFR1. Recombinant GST (Control) and GST-tagged domain D1 of the FGFR1 (GST-D1) were bound to Glutathione Sepharose and incubated with scFv proteins. Proteins bound to GST and GST-D1 were eluted and analyzed by Western blotting using specific antibodies. Cropped blots were displayed, full size blots are included in Supplementary Information.

    Journal: Scientific Reports

    Article Title: Antibody-induced dimerization of FGFR1 promotes receptor endocytosis independently of its kinase activity

    doi: 10.1038/s41598-017-07479-z

    Figure Lengend Snippet: Antibody fragments bind to the domain D1 of FGFR1. ( a ) Schematic representation of anti-FGFR1 antibody fragments used in this study. The scFv antibody format contains single antigen binding site formed by variable domains of heavy and light antibody chains (VH and VL), whereas scFv-Fc antibody fragments contain two identical binding sites for antigen fused by constant domains of heavy chain of human IgG1 (CH2 and CH3). ( b ) Analyzed antibodies recognize epitopes within domain D1 of FGFR1. scFvD2.myc was bound to the anti-c-Myc agarose and incubated with either purified full length extracellular part of FGFR1 fused with Fc fragment (FGFR1 D1-D2-D3-Fc) or with the Fc-fusion of the extracellular part of FGFR1 lacking domain D1 (FGFR1 D2-D3-Fc). Proteins bound to scFvD2.myc were analyzed with anti-Fc antibodies. ( c ) Direct Interaction of scFv’s with the domain D1 of the FGFR1. Recombinant GST (Control) and GST-tagged domain D1 of the FGFR1 (GST-D1) were bound to Glutathione Sepharose and incubated with scFv proteins. Proteins bound to GST and GST-D1 were eluted and analyzed by Western blotting using specific antibodies. Cropped blots were displayed, full size blots are included in Supplementary Information.

    Article Snippet: Protein A Sepharose and Glutathione Sepharose resins were from GE Healthcare (Piscataway, NJ, USA).

    Techniques: Binding Assay, Incubation, Purification, Recombinant, Western Blot