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soluble gst fusion  (Cytiva Europe)


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    Structured Review

    Cytiva Europe soluble gst fusion
    (A) Domain structure of the <t>human</t> <t>NEMO</t> protein, showing the two coiled coil domains (CC1 and CC2), the NEMO ubiquitin binding domain (NUB), leucine zipper (LZ) and zinc finger (ZF). (B) Immunoblot detection of biotinylated NEMO following purification of <t>GST-NEMO,</t> cleavage of the GST tag and biotinylation. Biotinylated NEMO was detected with streptavidin-alkaline phosphatase conjugate. (C) Example hits obtained from the array, compared to the same spot positions on negative control array. (D) Frequency histogram for the NEMO-probed protein microarray showing the range of Z-scores obtained. Protein interactors with a Z -score greater than three (Z>3; P<0.002) were deemed significant. Scores obtained for the canonical NEMO interactors IKKalpha (Z = 6.52) and IKKbeta (Z = 8.41) are shown for reference. Scores were calculated using Invitrogen Protoarray Prospector version 5.1 software. See for gene descriptions.
    Soluble Gst Fusion, supplied by Cytiva Europe, used in various techniques. Bioz Stars score: 96/100, based on 15195 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/ge+array+expression+analysis+suite+software/Fusion/pmc02808332-232-6-22
    Average 96 stars, based on 15195 article reviews
    soluble gst fusion - by Bioz Stars, 2026-09
    96/100 stars

    Images

    1) Product Images from "Expanding the Substantial Interactome of NEMO Using Protein Microarrays"

    Article Title: Expanding the Substantial Interactome of NEMO Using Protein Microarrays

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0008799

    (A) Domain structure of the human NEMO protein, showing the two coiled coil domains (CC1 and CC2), the NEMO ubiquitin binding domain (NUB), leucine zipper (LZ) and zinc finger (ZF). (B) Immunoblot detection of biotinylated NEMO following purification of GST-NEMO, cleavage of the GST tag and biotinylation. Biotinylated NEMO was detected with streptavidin-alkaline phosphatase conjugate. (C) Example hits obtained from the array, compared to the same spot positions on negative control array. (D) Frequency histogram for the NEMO-probed protein microarray showing the range of Z-scores obtained. Protein interactors with a Z -score greater than three (Z>3; P<0.002) were deemed significant. Scores obtained for the canonical NEMO interactors IKKalpha (Z = 6.52) and IKKbeta (Z = 8.41) are shown for reference. Scores were calculated using Invitrogen Protoarray Prospector version 5.1 software. See for gene descriptions.
    Figure Legend Snippet: (A) Domain structure of the human NEMO protein, showing the two coiled coil domains (CC1 and CC2), the NEMO ubiquitin binding domain (NUB), leucine zipper (LZ) and zinc finger (ZF). (B) Immunoblot detection of biotinylated NEMO following purification of GST-NEMO, cleavage of the GST tag and biotinylation. Biotinylated NEMO was detected with streptavidin-alkaline phosphatase conjugate. (C) Example hits obtained from the array, compared to the same spot positions on negative control array. (D) Frequency histogram for the NEMO-probed protein microarray showing the range of Z-scores obtained. Protein interactors with a Z -score greater than three (Z>3; P<0.002) were deemed significant. Scores obtained for the canonical NEMO interactors IKKalpha (Z = 6.52) and IKKbeta (Z = 8.41) are shown for reference. Scores were calculated using Invitrogen Protoarray Prospector version 5.1 software. See for gene descriptions.

    Techniques Used: Binding Assay, Western Blot, Purification, Negative Control, Microarray, Software

    (A) Immunoblot analysis of GST and GST-NEMO proteins used as control and bait for the pulldown assay. Proteins were detected using anti-GST/HRP conjugate following SDS-PAGE and membrane transfer. (B) Results of GST pulldown assays showing binding of NEMO to putative interactors identified by protein array screening. Each of the interactors and IKKbeta, a known NEMO binder, were overexpressed in transiently transfected HEK-293T cells and the resulting lysates applied to immobilized GST or GST-NEMO. Following incubation and washing, the samples were resolved by SDS-PAGE and the proteins detected using appropriate antibodies. Input lanes were loaded with 5–10% of HEK-293T lysates to confirm protein expression. The size of relevant protein markers is shown beside the blot image. (C–H) Coimmunoprecipitation assays between NEMO and putative binders in HEK-293T cells. Plasmids encoding Xpress-tagged NEMO or the empty parent vector and tagged putative binders were used to transfect HEK-293T cells and the resulting cell lysates used for coimmunoprecipitation assays. For each putative binder, immunoblots are shown for detection of the binder using a tag- or protein-specific antibody, and for detection of Xpress-tagged NEMO. For IKKbeta and each of the five putative interactors, substantial coimmunoprecipitation occurred only in the presence immunoprecipitated NEMO. Input lanes contained 5–10% of the precleared input volume used prior to addition of anti-Xpress antibody. Binding and washing steps were performed in the presence of 0.5% NP-40 for all proteins except SAG, where 0.1% NP-40 was used. (I) NEMO interacts with CALB1, CDK2, SAG, SENP2 and SYT1 in a mammalian two-hybrid system. Empty two-hybrid vectors were cotransfected as a negative control. The MyoD/Id and NEMO/IkappaBalpha protein pairs were used as positive controls, while putative interaction partners cotransfected with empty complementing vector were used as negative controls. For each pair tested, a significant increase (n = 6; two-tailed T test; P≤0.05) in luciferase activity was obtained in partner/NEMO experiments compared to partner/vector experiments (indicated by asterisks).
    Figure Legend Snippet: (A) Immunoblot analysis of GST and GST-NEMO proteins used as control and bait for the pulldown assay. Proteins were detected using anti-GST/HRP conjugate following SDS-PAGE and membrane transfer. (B) Results of GST pulldown assays showing binding of NEMO to putative interactors identified by protein array screening. Each of the interactors and IKKbeta, a known NEMO binder, were overexpressed in transiently transfected HEK-293T cells and the resulting lysates applied to immobilized GST or GST-NEMO. Following incubation and washing, the samples were resolved by SDS-PAGE and the proteins detected using appropriate antibodies. Input lanes were loaded with 5–10% of HEK-293T lysates to confirm protein expression. The size of relevant protein markers is shown beside the blot image. (C–H) Coimmunoprecipitation assays between NEMO and putative binders in HEK-293T cells. Plasmids encoding Xpress-tagged NEMO or the empty parent vector and tagged putative binders were used to transfect HEK-293T cells and the resulting cell lysates used for coimmunoprecipitation assays. For each putative binder, immunoblots are shown for detection of the binder using a tag- or protein-specific antibody, and for detection of Xpress-tagged NEMO. For IKKbeta and each of the five putative interactors, substantial coimmunoprecipitation occurred only in the presence immunoprecipitated NEMO. Input lanes contained 5–10% of the precleared input volume used prior to addition of anti-Xpress antibody. Binding and washing steps were performed in the presence of 0.5% NP-40 for all proteins except SAG, where 0.1% NP-40 was used. (I) NEMO interacts with CALB1, CDK2, SAG, SENP2 and SYT1 in a mammalian two-hybrid system. Empty two-hybrid vectors were cotransfected as a negative control. The MyoD/Id and NEMO/IkappaBalpha protein pairs were used as positive controls, while putative interaction partners cotransfected with empty complementing vector were used as negative controls. For each pair tested, a significant increase (n = 6; two-tailed T test; P≤0.05) in luciferase activity was obtained in partner/NEMO experiments compared to partner/vector experiments (indicated by asterisks).

    Techniques Used: Western Blot, SDS Page, Binding Assay, Protein Array, Transfection, Incubation, Expressing, Plasmid Preparation, Immunoprecipitation, Negative Control, Two Tailed Test, Luciferase, Activity Assay

    Related Articles

    Purification:

    Article Title: ETHYLENE RESPONSE FACTORS 4.1/4.2 with an EAR motif repress anthocyanin biosynthesis in red-skinned pears
    Article Snippet: .. Next, PyERF4.1-MBP, PyERF4.2-MBP, and PyERF3-HIS fusion proteins were expressed in DE3 Escherichia coli cells, and PyERF3 - HIS was purified using Ni Sepharose 6 Fast Flow (17-5318-06, GE Healthcare, Sweden). .. The collected elution extract was blotted with MBP or HIS antibodies (Beyotime, Shanghai, China).

    Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack
    Article Snippet: Eluted proteins were buffer exchanged into PBS and concentrated to 2 mg ml −1 using Amicon Ultra-15 centrifugal filters (EMD Millipore) with a 30-kDa molecular weight cut-off (MWCO). .. Concentrated proteins were purified further by HPLC size-exclusion chromatography using the following columns: Superose 6 Increase 10/300 GL (for human MICA α3–ferritin and MICB α3–ferritin), Superdex 200 (for ferritin) or tandem Superose 6 Increase 10/300 GL (for macaque MICA/B α3–ferritin) (GE Health-care) with PBS (fusion proteins) or HBS (ferritin) as running buffer at a flow rate of 0.8 ml min −1 . ..

    Article Title: Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif‐mediated interactions
    Article Snippet: Thereafter, cells were centrifuged at 4°C to remove cell debris and the supernatant was filtered and loaded onto a Nickel Sepharose Fast Flow column (GE Healthcare) in the case of pSY10 constructs. .. The fusion protein was eluted using binding buffer with 250 mM imidazole and then further purified using size‐exclusion chromatography on a Hi load 16/60 Sephacryl S‐100 column (GE Healthcare) in the binding buffer with pH adjusted to 7.4. .. The fusion protein was then cleaved with PreScission (produced in house) protease overnight at 4°C followed by a second run on the size‐exclusion chromatography column to remove the NusA protein. in the case of pETM33 constructs, after removing cell debris the supernatant was loaded onto PierceTM Glutathione Agarose beads (Thermo Scientific), washed with wash buffer (50 mM Tris, 300 mM NaCl, pH 7.8) and eluted in GST elution buffer (50 mM Tris, 300 mM NaCl, 10 mM reduced glutathione, pH 7.8).

    High Performance Liquid Chromatography:

    Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack
    Article Snippet: Eluted proteins were buffer exchanged into PBS and concentrated to 2 mg ml −1 using Amicon Ultra-15 centrifugal filters (EMD Millipore) with a 30-kDa molecular weight cut-off (MWCO). .. Concentrated proteins were purified further by HPLC size-exclusion chromatography using the following columns: Superose 6 Increase 10/300 GL (for human MICA α3–ferritin and MICB α3–ferritin), Superdex 200 (for ferritin) or tandem Superose 6 Increase 10/300 GL (for macaque MICA/B α3–ferritin) (GE Health-care) with PBS (fusion proteins) or HBS (ferritin) as running buffer at a flow rate of 0.8 ml min −1 . ..

    Size-exclusion Chromatography:

    Article Title: A vaccine targeting resistant tumours by dual T cell plus NK cell attack
    Article Snippet: Eluted proteins were buffer exchanged into PBS and concentrated to 2 mg ml −1 using Amicon Ultra-15 centrifugal filters (EMD Millipore) with a 30-kDa molecular weight cut-off (MWCO). .. Concentrated proteins were purified further by HPLC size-exclusion chromatography using the following columns: Superose 6 Increase 10/300 GL (for human MICA α3–ferritin and MICB α3–ferritin), Superdex 200 (for ferritin) or tandem Superose 6 Increase 10/300 GL (for macaque MICA/B α3–ferritin) (GE Health-care) with PBS (fusion proteins) or HBS (ferritin) as running buffer at a flow rate of 0.8 ml min −1 . ..

    Article Title: Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif‐mediated interactions
    Article Snippet: Thereafter, cells were centrifuged at 4°C to remove cell debris and the supernatant was filtered and loaded onto a Nickel Sepharose Fast Flow column (GE Healthcare) in the case of pSY10 constructs. .. The fusion protein was eluted using binding buffer with 250 mM imidazole and then further purified using size‐exclusion chromatography on a Hi load 16/60 Sephacryl S‐100 column (GE Healthcare) in the binding buffer with pH adjusted to 7.4. .. The fusion protein was then cleaved with PreScission (produced in house) protease overnight at 4°C followed by a second run on the size‐exclusion chromatography column to remove the NusA protein. in the case of pETM33 constructs, after removing cell debris the supernatant was loaded onto PierceTM Glutathione Agarose beads (Thermo Scientific), washed with wash buffer (50 mM Tris, 300 mM NaCl, pH 7.8) and eluted in GST elution buffer (50 mM Tris, 300 mM NaCl, 10 mM reduced glutathione, pH 7.8).

    Binding Assay:

    Article Title: Evolution of affinity between p53 transactivation domain and MDM2 across the animal kingdom demonstrates high plasticity of motif‐mediated interactions
    Article Snippet: Thereafter, cells were centrifuged at 4°C to remove cell debris and the supernatant was filtered and loaded onto a Nickel Sepharose Fast Flow column (GE Healthcare) in the case of pSY10 constructs. .. The fusion protein was eluted using binding buffer with 250 mM imidazole and then further purified using size‐exclusion chromatography on a Hi load 16/60 Sephacryl S‐100 column (GE Healthcare) in the binding buffer with pH adjusted to 7.4. .. The fusion protein was then cleaved with PreScission (produced in house) protease overnight at 4°C followed by a second run on the size‐exclusion chromatography column to remove the NusA protein. in the case of pETM33 constructs, after removing cell debris the supernatant was loaded onto PierceTM Glutathione Agarose beads (Thermo Scientific), washed with wash buffer (50 mM Tris, 300 mM NaCl, pH 7.8) and eluted in GST elution buffer (50 mM Tris, 300 mM NaCl, 10 mM reduced glutathione, pH 7.8).

    Incubation:

    Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells
    Article Snippet: cDNAs encoding CCP20, CCP22, or their chimeras were amplified by PCR and subcloned into pGEX4T-1 (GE Healthcare) at the EcoRI/SalI sites. .. GST fusion proteins were induced in Escherichia coli BL21 cells by incubation with 0.1 mM IPTG at 25°C for 2 h. The cells were lysed by sonication and the supernatants were passed over a glutathione-Sepharose 4B (GE Healthcare) column. ..

    Sonication:

    Article Title: Polydom/SVEP1 binds to Tie1 and promotes migration of lymphatic endothelial cells
    Article Snippet: cDNAs encoding CCP20, CCP22, or their chimeras were amplified by PCR and subcloned into pGEX4T-1 (GE Healthcare) at the EcoRI/SalI sites. .. GST fusion proteins were induced in Escherichia coli BL21 cells by incubation with 0.1 mM IPTG at 25°C for 2 h. The cells were lysed by sonication and the supernatants were passed over a glutathione-Sepharose 4B (GE Healthcare) column. ..



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