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SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a <t>GSH-agarose</t> pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.
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1) Product Images from "GAPDH Mediates Nitrosylation of Nuclear Proteins"

Article Title: GAPDH Mediates Nitrosylation of Nuclear Proteins

Journal: Nature cell biology

doi: 10.1038/ncb2114

SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a GSH-agarose pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.
Figure Legend Snippet: SNO-GAPDH interacts with SIRT1 near its nitrosylated Cys150 residue ( a ) Endogenous co-immunoprecipitation of SIRT1 and GAPDH in HEK293 cells treated with NO donor. Cells were treated with 200 μM GSH or GSNO for 16 hr prior to lysis. ( b ) Nitrosylated GAPDH (SNO-GAPDH) binds directly to SIRT1 in vitro . GST or GST-GAPDH was pre-treated with 100 μM GSH or GSNO for 30 min at 37°C. After desalting, recombinant SIRT1 was added and binding assessed by a GSH-agarose pulldown assay. ( c ) A small peptide corresponding to the region of GAPDH that spans Cys150 (Peptide-C150) blocks the interaction between SNO-GAPDH and SIRT1. The assay was performed as in b . ( d ) Mutation of Thr152 of GAPDH abolishes binding to SIRT1. Twenty-four hours after transfection with wild-type HA-GAPDH or the indicated point mutants, HEK293 cells were treated with 200 μM GSH or GSNO for 16 hr. Cell lysates were immunoprecipitated with anti-SIRT1 antibody and analyzed by western blotting with anti-HA antibody. HA-GAPDH, HA-tagged GAPDH.

Techniques Used: Immunoprecipitation, Lysis, In Vitro, Recombinant, Binding Assay, Mutagenesis, Transfection, Western Blot

2) Product Images from "Human T-Lymphotropic Virus Type 1 p30II Regulates Gene Transcription by Binding CREB Binding Protein/p300"

Article Title: Human T-Lymphotropic Virus Type 1 p30II Regulates Gene Transcription by Binding CREB Binding Protein/p300

Journal: Journal of Virology

doi: 10.1128/JVI.75.20.9885-9895.2001

p30 II directly binds the KIX domain of CBP/p300 in vitro . (A) Schematic diagram of binding motifs and functional domains of CBP and p300. (B) Diagram representing the structure of GST-CBP- and GST-p300-expressing plasmids used in the GST pull-down assay. (C) GST fusion proteins containing defined residues of CBP and p300 were expressed and coupled to glutathione-agarose beads. After extensive washing, glutathione-agarose beads coupled with GST-CBP or GST-p300 fusion protein were incubated with 35 S-labeled p30 II synthesized by in vitro transcription and translation as described in Materials and Methods. 35 S-labeled p30 II pulled down by glutathione-agarose beads coupled with GST-CBP or GST-p300 fusion protein (upper panel) was visualized by autoradiography after being resolved in SDS-PAGE gels. Coomassie-stained SDS-PAGE gel for each of the corresponding GST-fusion proteins used in pull-down assay (upper panel). The major protein in each lane was of the expected molecular mass (lane 1, molecular mass marker) (lower panel).
Figure Legend Snippet: p30 II directly binds the KIX domain of CBP/p300 in vitro . (A) Schematic diagram of binding motifs and functional domains of CBP and p300. (B) Diagram representing the structure of GST-CBP- and GST-p300-expressing plasmids used in the GST pull-down assay. (C) GST fusion proteins containing defined residues of CBP and p300 were expressed and coupled to glutathione-agarose beads. After extensive washing, glutathione-agarose beads coupled with GST-CBP or GST-p300 fusion protein were incubated with 35 S-labeled p30 II synthesized by in vitro transcription and translation as described in Materials and Methods. 35 S-labeled p30 II pulled down by glutathione-agarose beads coupled with GST-CBP or GST-p300 fusion protein (upper panel) was visualized by autoradiography after being resolved in SDS-PAGE gels. Coomassie-stained SDS-PAGE gel for each of the corresponding GST-fusion proteins used in pull-down assay (upper panel). The major protein in each lane was of the expected molecular mass (lane 1, molecular mass marker) (lower panel).

Techniques Used: In Vitro, Binding Assay, Functional Assay, Expressing, Pull Down Assay, Incubation, Labeling, Synthesized, Autoradiography, SDS Page, Staining, Marker

3) Product Images from "Novel Putative Targets of N-ethylmaleimide Sensitive Fusion Protein (NSF) and ?/? Soluble NSF Attachment Proteins (SNAPs) Include the Pak-Binding Nucleotide Exchange Factor ?PIX"

Article Title: Novel Putative Targets of N-ethylmaleimide Sensitive Fusion Protein (NSF) and ?/? Soluble NSF Attachment Proteins (SNAPs) Include the Pak-Binding Nucleotide Exchange Factor ?PIX

Journal: Journal of cellular biochemistry

doi: 10.1002/jcb.20998

NSF interacts with the PAK binding exchange factor βPIX in vitro and in vivo. GST fusion proteins of isolated yeast two-hybrid βPIX clone (GST-βPIX-c) and the C-terminus of GluR2 (GST-GluR2-c) were immobilized on glutathione-sepharose
Figure Legend Snippet: NSF interacts with the PAK binding exchange factor βPIX in vitro and in vivo. GST fusion proteins of isolated yeast two-hybrid βPIX clone (GST-βPIX-c) and the C-terminus of GluR2 (GST-GluR2-c) were immobilized on glutathione-sepharose

Techniques Used: Binding Assay, In Vitro, In Vivo, Isolation

Related Articles

Centrifugation:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Affinity Chromatography:

Article Title: A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *
Article Snippet: .. GST fusion proteins were expressed in bacteria, purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences), and quantified as described ( ). ..

Purification:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
Article Snippet: .. For affinity purification of UNC-76 antisera, purified pGEX-H4 protein was cross-linked to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to established protocols ( ). .. Affinity purified UNC-76 antisera were used at a 1:100 dilution for immunostaining and a 1:1000 dilution for Western blots.

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function
Article Snippet: .. The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions. .. The recombinant protein was verified by SDS-PAGE and by immunoblotting with α-GST (Amersham) or α-GST/RG- polyclonal antibodies.

Article Title: A Docking Site in MKK4 Mediates High Affinity Binding to JNK MAPKs and Competes with Similar Docking Sites in JNK Substrates *
Article Snippet: .. GST fusion proteins were expressed in bacteria, purified by affinity chromatography using glutathione-Sepharose (Amersham Biosciences), and quantified as described ( ). ..

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
Article Snippet: .. GST-M6CK protein was purified from expressing BL21 bacteria using glutathione Sepharose (GE Healthcare) as described ( ). .. Purification of PRMT5, WDR77, and ICLN proteins was as described ( , ).

Expressing:

Article Title: Histone phosphorylation by TRPM6’s cleaved kinase attenuates adjacent arginine methylation to regulate gene expression
Article Snippet: .. GST-M6CK protein was purified from expressing BL21 bacteria using glutathione Sepharose (GE Healthcare) as described ( ). .. Purification of PRMT5, WDR77, and ICLN proteins was as described ( , ).

Sonication:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Affinity Purification:

Article Title: The Kinesin-associated Protein UNC-76 Is Required for Axonal Transport in the Drosophila Nervous System
Article Snippet: .. For affinity purification of UNC-76 antisera, purified pGEX-H4 protein was cross-linked to glutathione-Sepharose 4B (Amersham Biosciences, Piscataway, NJ) according to established protocols ( ). .. Affinity purified UNC-76 antisera were used at a 1:100 dilution for immunostaining and a 1:1000 dilution for Western blots.

Recombinant:

Article Title: The WD40 protein Morg1 facilitates Par6-aPKC binding to Crb3 for apical identity in epithelial cells
Article Snippet: .. In brief, recombinant proteins were expressed in the E. coli strain BL21, and cells were homogenized by sonication; after centrifugation, GST- or MBP-tagged proteins were purified using glutathione-Sepharose 4B (GE Healthcare) or amylose resin (New England Biolabs, Inc.), respectively. .. Recombinant Cdc42 (Q61L) and Cdc42 (T17N), both of which lack the C-terminal residues 189–191 and carry the C188S substitution for protein stabilization, were expressed as GST fusion protein in the E. coli strain BL21, and proteins were applied to glutathione-Sepharose 4B.

Article Title: Nuclear Factor 90, a cellular dsRNA binding protein inhibits the HIV Rev-export function
Article Snippet: .. The recombinant protein was purified using a glutathione-Sepharose 4B column (MicroSpin™ GST Purification Module, Amersham) following the manufacturer's instructions. .. The recombinant protein was verified by SDS-PAGE and by immunoblotting with α-GST (Amersham) or α-GST/RG- polyclonal antibodies.

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    GE Healthcare glutathione sepharose 4b
    Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or <t>GST-KSRP-Sepharose</t> beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P
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    Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P

    Journal: Journal of Cell Science

    Article Title: Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA

    doi: 10.1242/jcs.056176

    Figure Lengend Snippet: Dvl3-KSRP interaction is RNA dependent and Dvl3 complex harbors Ctnnb1 mRNA. ( A ) F9 cells were co-transfected with HA-Dvl3-GFP2 and FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-HA affinity matrix. Interaction of KSRP with exogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( B ) F9 cells were transfected with FLAG-KSRP for 24 hours followed by cell lysis. The lysates were then incubated without or with indicated amounts of RNaseA at room temperature for 10 minutes followed by affinity pull-downs with anti-Dvl3 antibodies. Interaction of KSRP with endogenous Dvl3 was probed by immunoblotting with anti-FLAG antibodies. ( C ) To test whether the in vitro interaction of KSRP and Dvl3 is also RNA dependent, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST or GST-KSRP-Sepharose beads in the absence or presence (5 μg) of either 3′-UTR of Ctnnb1 or Gapdh . The interaction was visualized by SDS-PAGE and autoradiography. ( D ) RNA immunoprecipitation assay was performed on F9 cell lysates with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by RT-PCR with primers specific for Ctnnb1 , Myc or Fzd7 . Representative gel of two independent experiments that proved highly reproducible is displayed. ( E ) F9 cells were treated with Wnt3a (10 ng/ml) for indicated periods of time and RNA immunoprecipitation assay was performed with either control mouse IgG or anti-Dvl3 antibodies. The RNA isolated from the immunoprecipitates was analyzed by quantitative real-time PCR with β-catenin specific primers. Representative blots of two independent experiments that proved highly reproducible were displayed. The data represent mean values ± s.e.m. from two independent experiments whose results were in high agreement. In the lower panel, a representative gel is displayed. ## P

    Article Snippet: Fusion polypeptides of GST and GST-KSRP were induced in Eschericia coli with 0.1 mM isopropyl-β-galactopyranoside at 30°C for 4 hours and affinity purified using 50% slurry of glutathione-Sepharose 4B (GE Life Sciences) according to the batch purification method suggested by the manufacturer.

    Techniques: Transfection, Lysis, Incubation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography, Immunoprecipitation, Isolation, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

    KSRP interacts with Dvl3. ( A ) F9 cells were transiently transfected with FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. Asterisks indicate the bands of immunoglobulin heavy and light chains. ( B ) F9 cells were treated with 100 nM of Dvl3 siRNA for 24 hours followed by transient expression of FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. ( C ) F9 cell lysates were immunoprecipitated with either rabbit control IgG or rabbit anti-KSRP polyclonal antibody and the interaction of KSRP with Dvl3 was visualized by probing the blots with anti-Dvl3 mouse monoclonal antibody. ( D ) F9 cells were transiently transfected with empty vector or FLAG-KSRP for 24 hours. The cells were then treated with Wnt3a (10 ng/ml) for indicated period of time followed by cell lysis and affinity pull-downs with anti-Dvl3 specific antibodies followed by immunoblotting with anti-FLAG antibodies. ( E ) To test the direct interaction of Dvl3 with KSRP, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST- or GST-KSRP-Sepharose beads in the presence of 0.8% BSA. The interaction was visualized by SDS-PAGE and autoradiography. Representative blots of three independent experiments that proved highly reproducible are shown. * P

    Journal: Journal of Cell Science

    Article Title: Dishevelled-KSRP complex regulates Wnt signaling through post-transcriptional stabilization of β-catenin mRNA

    doi: 10.1242/jcs.056176

    Figure Lengend Snippet: KSRP interacts with Dvl3. ( A ) F9 cells were transiently transfected with FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. Asterisks indicate the bands of immunoglobulin heavy and light chains. ( B ) F9 cells were treated with 100 nM of Dvl3 siRNA for 24 hours followed by transient expression of FLAG-KSRP for 24 hours followed by cell lysis and affinity pull-downs with either mouse control IgG or anti-Dvl3 mouse monoclonal antibody. Interaction of KSRP with Dvl3 was visualized by probing the blots with anti-FLAG antibody. ( C ) F9 cell lysates were immunoprecipitated with either rabbit control IgG or rabbit anti-KSRP polyclonal antibody and the interaction of KSRP with Dvl3 was visualized by probing the blots with anti-Dvl3 mouse monoclonal antibody. ( D ) F9 cells were transiently transfected with empty vector or FLAG-KSRP for 24 hours. The cells were then treated with Wnt3a (10 ng/ml) for indicated period of time followed by cell lysis and affinity pull-downs with anti-Dvl3 specific antibodies followed by immunoblotting with anti-FLAG antibodies. ( E ) To test the direct interaction of Dvl3 with KSRP, in vitro synthesized 35 S-labeled Dvl3 was used in pull-down experiments with either GST- or GST-KSRP-Sepharose beads in the presence of 0.8% BSA. The interaction was visualized by SDS-PAGE and autoradiography. Representative blots of three independent experiments that proved highly reproducible are shown. * P

    Article Snippet: Fusion polypeptides of GST and GST-KSRP were induced in Eschericia coli with 0.1 mM isopropyl-β-galactopyranoside at 30°C for 4 hours and affinity purified using 50% slurry of glutathione-Sepharose 4B (GE Life Sciences) according to the batch purification method suggested by the manufacturer.

    Techniques: Transfection, Lysis, Expressing, Immunoprecipitation, Plasmid Preparation, In Vitro, Synthesized, Labeling, SDS Page, Autoradiography

    In Vitro Pull-Down Analysis of the Stromal Regions of MCD1 and ARC6. (A) Recombinant His-ARC6 N binds to GST-MCD1 C or GST-MCD1 C(∆277-314) . Glutathione-Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST-tagged MCD1 C (lane 4), MCD1C (∆277-314) (lane 5), or MCD1 N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His-ARC6 N . Proteins were eluted and analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Recombinant MBP-PARC6 N -His or His-ARC6 C was not precipitated from crude E. coli extracts by Glutathione-Sepharose 4B beads coated with GST-MCD1 C . All assays were performed more than three times.

    Journal: The Plant Cell

    Article Title: MCD1 Associates with FtsZ Filaments via the Membrane-Tethering Protein ARC6 to Guide Chloroplast Division

    doi: 10.1105/tpc.18.00189

    Figure Lengend Snippet: In Vitro Pull-Down Analysis of the Stromal Regions of MCD1 and ARC6. (A) Recombinant His-ARC6 N binds to GST-MCD1 C or GST-MCD1 C(∆277-314) . Glutathione-Sepharose 4B beads were treated with buffer only (lane 2) or coated with GST (lane 3), GST-tagged MCD1 C (lane 4), MCD1C (∆277-314) (lane 5), or MCD1 N (lane 6). The beads were then incubated with crude extracts of E. coli cells expressing His-ARC6 N . Proteins were eluted and analyzed by immunoblotting with anti-His and anti-GST antibodies. (B) Recombinant MBP-PARC6 N -His or His-ARC6 C was not precipitated from crude E. coli extracts by Glutathione-Sepharose 4B beads coated with GST-MCD1 C . All assays were performed more than three times.

    Article Snippet: For the GST pull-down assays, 50 μL samples of a 50% slurry of Glutathione-Sepharose 4B beads (GE Healthcare) were equilibrated in LT buffer (lysis buffer including 0.1% Triton X-100).

    Techniques: In Vitro, Recombinant, Incubation, Expressing

    Expression and purification of GST-GFP, GST-TgCaMKrk, and GST-TgCaMKrkKA. Proteins were expressed by using a wheat germ cell-free protein synthesis system. Total wheat germ extracts were subjected to affinity chromatography using glutathione-Sepharose beads. The purified proteins were separated on denaturing gels and then ( a ) silver stained or ( b ) transferred to a nitrocellulose sheet and probed with anti-GST antibodies. Lanes 1 and 2, GST-GFP; Lanes 3 and 4, GST-TgCaMKrk; Lanes 5 and 6, GST-TgCaMKrkKA. Lanes 1, 3, and 5, total wheat germ extracts; Lanes 2, 4, and 6, purified proteins. Molecular masses (kDa) are shown on the left

    Journal: Parasites & Vectors

    Article Title: Characterization of a Toxoplasma gondii calcium calmodulin-dependent protein kinase homolog

    doi: 10.1186/s13071-016-1676-1

    Figure Lengend Snippet: Expression and purification of GST-GFP, GST-TgCaMKrk, and GST-TgCaMKrkKA. Proteins were expressed by using a wheat germ cell-free protein synthesis system. Total wheat germ extracts were subjected to affinity chromatography using glutathione-Sepharose beads. The purified proteins were separated on denaturing gels and then ( a ) silver stained or ( b ) transferred to a nitrocellulose sheet and probed with anti-GST antibodies. Lanes 1 and 2, GST-GFP; Lanes 3 and 4, GST-TgCaMKrk; Lanes 5 and 6, GST-TgCaMKrkKA. Lanes 1, 3, and 5, total wheat germ extracts; Lanes 2, 4, and 6, purified proteins. Molecular masses (kDa) are shown on the left

    Article Snippet: Purification of recombinant proteins Wheat germ extracts were mixed with 10 μl of a 50 % slurry of glutathione-Sepharose beads (GE Healthcare, Little Chalfont, Buckinghamshire, UK) for 16 h. The beads were then washed three times with phosphate-buffered saline (PBS).

    Techniques: Expressing, Purification, Affinity Chromatography, Staining

    (A) Arkadia ubiquitinates poly-SUMO2 chains in a SIM- and RING-dependent manner. HEK293 cells were transfected with either Flag empty vector, Flag-Ark-wt, Flag-Ark-RING*, Flag-Ark-SIM123*, or Flag-RNF4 as a positive control. The different Flag constructs were purified on anti-Flag agarose beads, and 10 μl of Flag-purified proteins was incubated in the presence of recombinant poly-SUMO2 chains for 30 min on ice. In vitro ubiquitination was then performed for 45 min at 37°C in the presence of HA-Ub, E1 (UBE1), E2 (UbcH5b and UbcH5c), and ATP. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. The relative level of Flag-purified proteins used in the assay was evaluated in parallel by Western blotting using an anti-Flag antibody, as shown in the lower blot. (B) Arkadia and RNF4 independently ubiquitinate poly-SUMO2 chains. HEK293 cells were transfected with siArk or siRNF4. At 48 h after siRNA transfection, cells were transfected with Flag-Ark-wt or Flag-RNF4-wt, as indicated. At 24 h after plasmid transfection, cell lysates were extracted and Arkadia and RNF4 expression were evaluated by Western blotting using anti-Ark and anti-RNF4 as well as antiactin as a loading control. The rest of the lysate was purified with anti-Flag agarose beads. Ten microliters of Flag-purified proteins was used for in vitro ubiquitination assay of poly-SUMO2 chains, as described for panel A. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. Numbers to the left of the gels are molecular masses (in kDa).

    Journal: Molecular and Cellular Biology

    Article Title: Arkadia, a Novel SUMO-Targeted Ubiquitin Ligase Involved in PML Degradation

    doi: 10.1128/MCB.01019-12

    Figure Lengend Snippet: (A) Arkadia ubiquitinates poly-SUMO2 chains in a SIM- and RING-dependent manner. HEK293 cells were transfected with either Flag empty vector, Flag-Ark-wt, Flag-Ark-RING*, Flag-Ark-SIM123*, or Flag-RNF4 as a positive control. The different Flag constructs were purified on anti-Flag agarose beads, and 10 μl of Flag-purified proteins was incubated in the presence of recombinant poly-SUMO2 chains for 30 min on ice. In vitro ubiquitination was then performed for 45 min at 37°C in the presence of HA-Ub, E1 (UBE1), E2 (UbcH5b and UbcH5c), and ATP. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. The relative level of Flag-purified proteins used in the assay was evaluated in parallel by Western blotting using an anti-Flag antibody, as shown in the lower blot. (B) Arkadia and RNF4 independently ubiquitinate poly-SUMO2 chains. HEK293 cells were transfected with siArk or siRNF4. At 48 h after siRNA transfection, cells were transfected with Flag-Ark-wt or Flag-RNF4-wt, as indicated. At 24 h after plasmid transfection, cell lysates were extracted and Arkadia and RNF4 expression were evaluated by Western blotting using anti-Ark and anti-RNF4 as well as antiactin as a loading control. The rest of the lysate was purified with anti-Flag agarose beads. Ten microliters of Flag-purified proteins was used for in vitro ubiquitination assay of poly-SUMO2 chains, as described for panel A. Ubiquitinated poly-SUMO2 was visualized by Western blotting as a shift of higher-molecular-mass species using anti-SUMO2/3 antibody. Numbers to the left of the gels are molecular masses (in kDa).

    Article Snippet: For GST pulldown, 5 μg of each GST protein was conjugated to 20 μl of a glutathione-Sepharose bead slurry (GE Healthcare), and the conjugated proteins were incubated at 4°C overnight on a wheel with HEK293 cell lysate or with 1 μg of recombinant poly-SUMO2 chains (Boston Biochem) in lysis buffer (20 mM Tris, pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% NP-40, 10% glycerol, 20 mM NEM, protease inhibitors [Roche]).

    Techniques: Transfection, Plasmid Preparation, Positive Control, Construct, Purification, Incubation, Recombinant, In Vitro, Western Blot, Expressing, Ubiquitin Assay