streptavidin agarose conjugated beads  (Millipore)


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    Structured Review

    Millipore streptavidin agarose conjugated beads
    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with <t>streptavidin–agarose</t> beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
    Streptavidin Agarose Conjugated Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 88/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin agarose conjugated beads/product/Millipore
    Average 88 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    streptavidin agarose conjugated beads - by Bioz Stars, 2020-04
    88/100 stars

    Images

    1) Product Images from "Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors"

    Article Title: Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors

    Journal: EMBO Molecular Medicine

    doi: 10.1002/emmm.201100124

    Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with streptavidin–agarose beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.
    Figure Legend Snippet: Rescue of CMG2 cell surface expression and function in HFS patient cells upon exposure to proteasome inhibitors Patient-derived fibroblasts were incubated with 10 µM of BZ or not for 16 h. CMG2 was immunoprecipitated using the goat anti-human CMG2 antibody from 300 µg of cell lysates and analysed by SDS–PAGE under non-reducing conditions and Western blotting using the 2F6 anti-hCMG2 antibody. Fibroblasts derived from Patients 1, 2 and 5 and control cells were incubated with 10 µM BZ or not for 16 h and subsequently incubated with 0.2 mg/ml NHS-SS-biotin. Cell lysates (300 µg of total proteins) were subjected to immunoprecipitation with streptavidin–agarose beads and analysed by SDS–PAGE and Western blotting probed with anti-hCMG2, anti-calnexin and anti-hTransferin receptor antibodies. * indicates unspecific band recognized by the 2F6 antibody after surface biotinylation and immunoprecipitation. This band is also observed for Patient 4 fibroblasts that express undetectable levels of CMG2. While calnexin (Cnx) was absent in these immunoprecipitations, it was readily detected in cell extracts (Supplementary Fig 4E ). Under non-reducing conditions, human transferrin receptor (hTfR) migrates as a covalent dimer. Fibroblasts derived from Patient 1 and control cells were incubated with 10 µM BZ for 16 h and subsequently incubated with anthrax PA at a 500 ng/mL final concentration. After different time points, cell lysates were immunoprecipitated with an anti-hCMG2 antibody. Samples were analysed by SDS–PAGE under reducing or non-reducing conditions and Western blotting with an anti-hCMG2, an anti-ubiquitin, and anti-PA antibodies.

    Techniques Used: Expressing, Derivative Assay, Incubation, Immunoprecipitation, SDS Page, Western Blot, Concentration Assay

    2) Product Images from "Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8"

    Article Title: Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8

    Journal: PLoS ONE

    doi: 10.1371/journal.pone.0119864

    Non-glycosylated TEM8 is an ER quality control and ERAD substrate. A) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated or not with MG132, an inhibitor of the proteasome or Bafilomycin A1, a drug preventing endosomal acidification and thus lysosomal degradation. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and TEM8-HA. B) HEK cells stably expressing CMG2 under the control of a tetracycline inducible promotor were induced for 24h with 0.1μg/ml doxycycline. Cells were treated or not with MG132 or Bafilomycin A1. Immunoprecipitates against CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and CMG2-V5. C) HeLa cells were treated or not with tunicamycin, an antibiotic blocking the co-translational transfer of glycan sidechains in the ER by blocking the oligosaccharyltransferase (OST) for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for TEM8 or Calnexin as a negative control. D) RpeI cells were treated or not with tunicamycin for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for CMG2 or Calnexin as a negative control.
    Figure Legend Snippet: Non-glycosylated TEM8 is an ER quality control and ERAD substrate. A) HeLa cells were transfected for 48h with the respective cDNAs. Cells were treated or not with MG132, an inhibitor of the proteasome or Bafilomycin A1, a drug preventing endosomal acidification and thus lysosomal degradation. Immunoprecipitates against TEM8-HA were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and TEM8-HA. B) HEK cells stably expressing CMG2 under the control of a tetracycline inducible promotor were induced for 24h with 0.1μg/ml doxycycline. Cells were treated or not with MG132 or Bafilomycin A1. Immunoprecipitates against CMG2-V5 were analyzed by SDS-PAGE and Western Blotting against Ubiquitin and CMG2-V5. C) HeLa cells were treated or not with tunicamycin, an antibiotic blocking the co-translational transfer of glycan sidechains in the ER by blocking the oligosaccharyltransferase (OST) for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for TEM8 or Calnexin as a negative control. D) RpeI cells were treated or not with tunicamycin for 16h. Surface proteins were labeled with biotin and immunoprecipitates against streptavidin were analysed for CMG2 or Calnexin as a negative control.

    Techniques Used: Transfection, SDS Page, Western Blot, Stable Transfection, Expressing, Blocking Assay, Labeling, Negative Control

    Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2. A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 μg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5.
    Figure Legend Snippet: Number and localization of glycan sidechains determine trafficking efficiency of TEM8 and CMG2. A) Endoglycosidase H (EndoH) treatment on TEM8 and CMG2 single mutants. HeLa cells were transfected for 48h with the respective cDNAs. 40 μg of cell extracts were treated or not with EndoH as described before. Samples were analyzed by SDS-PAGE and Western Blotting. B) Quantification of surface biotinylation experiments to determine amount of TEM8 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 C) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against TEM8-HA. D) Quantification of surface biotinylation experiments to determine amount of CMG2 at the cell surface. All mutants were corrected for their expression levels and then normalized to WT, which was set at 100%. Errors represent standard deviation. Statistics were calculated using an unpaired t-test. n ≥ 3. * p≤0.05, ** p≤0.01, *** p≤0.001 E) Representative Western Blots of surface biotinylation. HeLa cells were transfected 48h with the respective cDNAs. Proteins at the cell surface were labeled with biotin, immunoprecipitated with streptavidin beads and blotted against CMG2-V5.

    Techniques Used: Transfection, SDS Page, Western Blot, Expressing, Standard Deviation, Labeling, Immunoprecipitation

    Related Articles

    DNA Extraction:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Paragraph title: DNA extraction and sequence validation of guide RNA edits ... Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS.

    Transfection:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Cells were transfected with SBPempty vector (control cells) and SBP-IFITM1 for 48 h. 48 h after transfection, cells were treated with carrier or with 100 ng/ml IFNγ (Invitrogen) for 24 h in order to "stabilize" potential interferon-activated SBP-IFITM1 interacting proteins. .. Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS.

    Methylation:

    Article Title: Mannose Trimming Is Required for Delivery of a Glycoprotein from EDEM1 to XTP3-B and to Late Endoplasmic Reticulum-associated Degradation Steps *
    Article Snippet: Rainbow 14 C-labeled methylated protein standards were obtained from GE Healthcare. .. Streptavidin-agarose-conjugated beads and other common reagents were from Sigma.

    Mutagenesis:

    Article Title: Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8
    Article Snippet: Mutations were generated using the QuikChange II XL Site-Directed Mutagenesis Kit (Agilent, Santa Clara, CA). .. Streptavidin-agarose conjugated beads were from Sigma-Aldrich (St. Louis, MO), protein G beads were from GE Healthcare (Uppsala, Sweden) and HA-beads from Roche (Basel, Switzerland); Treatments with N-glycosidase and Endoglycosidase H (New England Biolabs, Ipswich, MA) were performed as previously described [ ].

    Protease Inhibitor:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Cells were washed twice in ice cold PBS and scraped into buffer containing 100 mM KCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na 3 VO 4 , 10 mM NaF, 10% (v/v) glycerol, protease inhibitor mix, and 0.1% triton X-100, then incubated for 30 min on ice and centrifuged at 13,000 rpm for 15 min at 4°C. .. Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS.

    Incubation:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Cells were washed twice in ice cold PBS and scraped into buffer containing 100 mM KCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 1 mM EGTA, 0.5 mM Na 3 VO 4 , 10 mM NaF, 10% (v/v) glycerol, protease inhibitor mix, and 0.1% triton X-100, then incubated for 30 min on ice and centrifuged at 13,000 rpm for 15 min at 4°C. .. Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS.

    Labeling:

    Article Title: Mannose Trimming Is Required for Delivery of a Glycoprotein from EDEM1 to XTP3-B and to Late Endoplasmic Reticulum-associated Degradation Steps *
    Article Snippet: Promix cell labeling mix ([35 S]Met plus [35 S]Cys) > 1000 Ci/mmol was from PerkinElmer Life Sciences. .. Streptavidin-agarose-conjugated beads and other common reagents were from Sigma.

    Purification:

    Article Title: Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8
    Article Snippet: Anthrax toxin was purified as described before [ ]. .. Streptavidin-agarose conjugated beads were from Sigma-Aldrich (St. Louis, MO), protein G beads were from GE Healthcare (Uppsala, Sweden) and HA-beads from Roche (Basel, Switzerland); Treatments with N-glycosidase and Endoglycosidase H (New England Biolabs, Ipswich, MA) were performed as previously described [ ].

    Produced:

    Article Title: Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8
    Article Snippet: Polyclonal goat antibody (#771B) against Protective Antigen (PA) was from List Biological Laboratories (Campbell, CA) and used at 1/2000 dilution, monoclonal mouse V5 antibody (#R960–25) was from Invitrogen (Carlsbad, CA) and used at 1/2000 dilution; monoclonal rat HA-HRP antibody (#12 013 819 001) was from Roche (Basel, Switzerland) and used at 1/2000 dilution; monoclonal mouse Ubiquitin antibody (#sc-8017) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and used at 1/500 dilution; polyclonal rabbit calnexin antibody was produced by Eurogentec for our lab [ ] and used at 1/2000 dilution; polyclonal rabbit BiP antibody (#ab21685) from Abcam (Cambridge, UK) used at 1/1000 dilution; polyclonal rabbit antibody against TEM8 was generated in our lab and used at 1/2000 and polyclonal goat CMG2 antibody (#AF2940) was from R & D Systems (Minneapolis, MN) and used at 1/2000 dilution. .. Streptavidin-agarose conjugated beads were from Sigma-Aldrich (St. Louis, MO), protein G beads were from GE Healthcare (Uppsala, Sweden) and HA-beads from Roche (Basel, Switzerland); Treatments with N-glycosidase and Endoglycosidase H (New England Biolabs, Ipswich, MA) were performed as previously described [ ].

    Concentration Assay:

    Article Title: Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors
    Article Snippet: Reagents, proteasome inhibition and EndoH treatment The anti-human CMG2 monoclonal antibody 2F6 was obtained by genetic immunization of rats with an hCMG2 construct (Genovac, Germany); polyclonal goat anti human CMG2 antibodies were from R & D (Ref. AF2940), PA of anthrax toxin, anti-PA and anti-CMG2 antibodies were provided from the Leppla laboratory; goat anti-human CMG2 antibody was from R & D Systems; anti-HA-HRP antibody, anti-HA beads were from Roche (Basel, Switzerland); anti human-transferin receptor from Invitrogen (Carlsbad, CA); protein G-agarose-conjugated beads were from GE Healthcare; anti-V5 antibody was from Invitrogen; streptavidin–agarose conjugated beads from Sigma–Aldrich (St. Louis, MO); anti-ubiquitin antibody from Santa Cruz; HRP secondary antibodies from Pierce Chemical Co. (Rockford, IL); Alexa-conjugated secondary antibodies from Jackson Immunoresearch. .. The proteasome inhibitors—MG132 was from Sigma, BZ—were used at a final concentration of 10 µM for 16 h in complete medium.

    Generated:

    Article Title: Differential Dependence on N-Glycosylation of Anthrax Toxin Receptors CMG2 and TEM8
    Article Snippet: Polyclonal goat antibody (#771B) against Protective Antigen (PA) was from List Biological Laboratories (Campbell, CA) and used at 1/2000 dilution, monoclonal mouse V5 antibody (#R960–25) was from Invitrogen (Carlsbad, CA) and used at 1/2000 dilution; monoclonal rat HA-HRP antibody (#12 013 819 001) was from Roche (Basel, Switzerland) and used at 1/2000 dilution; monoclonal mouse Ubiquitin antibody (#sc-8017) from Santa Cruz Biotechnology Inc. (Santa Cruz, CA) and used at 1/500 dilution; polyclonal rabbit calnexin antibody was produced by Eurogentec for our lab [ ] and used at 1/2000 dilution; polyclonal rabbit BiP antibody (#ab21685) from Abcam (Cambridge, UK) used at 1/1000 dilution; polyclonal rabbit antibody against TEM8 was generated in our lab and used at 1/2000 and polyclonal goat CMG2 antibody (#AF2940) was from R & D Systems (Minneapolis, MN) and used at 1/2000 dilution. .. Streptavidin-agarose conjugated beads were from Sigma-Aldrich (St. Louis, MO), protein G beads were from GE Healthcare (Uppsala, Sweden) and HA-beads from Roche (Basel, Switzerland); Treatments with N-glycosidase and Endoglycosidase H (New England Biolabs, Ipswich, MA) were performed as previously described [ ].

    Inhibition:

    Article Title: Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors
    Article Snippet: .. Reagents, proteasome inhibition and EndoH treatment The anti-human CMG2 monoclonal antibody 2F6 was obtained by genetic immunization of rats with an hCMG2 construct (Genovac, Germany); polyclonal goat anti human CMG2 antibodies were from R & D (Ref. AF2940), PA of anthrax toxin, anti-PA and anti-CMG2 antibodies were provided from the Leppla laboratory; goat anti-human CMG2 antibody was from R & D Systems; anti-HA-HRP antibody, anti-HA beads were from Roche (Basel, Switzerland); anti human-transferin receptor from Invitrogen (Carlsbad, CA); protein G-agarose-conjugated beads were from GE Healthcare; anti-V5 antibody was from Invitrogen; streptavidin–agarose conjugated beads from Sigma–Aldrich (St. Louis, MO); anti-ubiquitin antibody from Santa Cruz; HRP secondary antibodies from Pierce Chemical Co. (Rockford, IL); Alexa-conjugated secondary antibodies from Jackson Immunoresearch. .. The proteasome inhibitors—MG132 was from Sigma, BZ—were used at a final concentration of 10 µM for 16 h in complete medium.

    Construct:

    Article Title: Hyaline Fibromatosis Syndrome inducing mutations in the ectodomain of anthrax toxin receptor 2 can be rescued by proteasome inhibitors
    Article Snippet: .. Reagents, proteasome inhibition and EndoH treatment The anti-human CMG2 monoclonal antibody 2F6 was obtained by genetic immunization of rats with an hCMG2 construct (Genovac, Germany); polyclonal goat anti human CMG2 antibodies were from R & D (Ref. AF2940), PA of anthrax toxin, anti-PA and anti-CMG2 antibodies were provided from the Leppla laboratory; goat anti-human CMG2 antibody was from R & D Systems; anti-HA-HRP antibody, anti-HA beads were from Roche (Basel, Switzerland); anti human-transferin receptor from Invitrogen (Carlsbad, CA); protein G-agarose-conjugated beads were from GE Healthcare; anti-V5 antibody was from Invitrogen; streptavidin–agarose conjugated beads from Sigma–Aldrich (St. Louis, MO); anti-ubiquitin antibody from Santa Cruz; HRP secondary antibodies from Pierce Chemical Co. (Rockford, IL); Alexa-conjugated secondary antibodies from Jackson Immunoresearch. .. The proteasome inhibitors—MG132 was from Sigma, BZ—were used at a final concentration of 10 µM for 16 h in complete medium.

    Sequencing:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Paragraph title: DNA extraction and sequence validation of guide RNA edits ... Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS.

    Binding Assay:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS. .. Binding proteins were eluted with a buffer containing 20 mM HEPES pH 8, 2 mM DTT, and 8 M Urea.

    Plasmid Preparation:

    Article Title: The effects of IFITM1 and IFITM3 gene deletion on IFNγ stimulated protein synthesis.
    Article Snippet: Cells were transfected with SBPempty vector (control cells) and SBP-IFITM1 for 48 h. 48 h after transfection, cells were treated with carrier or with 100 ng/ml IFNγ (Invitrogen) for 24 h in order to "stabilize" potential interferon-activated SBP-IFITM1 interacting proteins. .. Streptavidin Agarose conjugated beads (Millipore) were prewashed with in PBS.

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    Millipore streptavidin conjugated agarose beads
    CARM1 is in association with Sox2. (A) Co-immunoprecipitation of Sox2 with CARM1. Whole cell extracts (WCEs) of MCF7 cells transfected with (+) or without (-) FLAG-CARM1 or GFP-Sox2, were subjected to immunoprecipitation with anti-FLAG antibody and blotted with antibodies against GFP or FLAG. (B) Co-precipitation of endogenous CARM1 with Sox2 by DNA pull-down. Biotin-labeled probes covering Sox2-binding sites in fgf4 and utf1 respectively were incubated with WCEs of P19 cells and <t>streptavidin-conjugated</t> agarose beads. The proteins precipitated with beads were denatured and resolved in SDS-PAGE and detected by immunoblotting with antibodies against Sox2 or CARM1. NC: negative control, no probe added. (C) Schematic drawing of the wild type and truncated CARM1 fragments. (D) GST pull-down assays to detect the interaction of Sox2 with CARM1 derivates. GST or GST-Sox2 was first incubated with WCEs of HEK293T cells ectopically expressing FLAG-CARM1 derivates. The GST pull-down products were immunoblotted with anti-FLAG antibody. Input: WCE control. (E) Schematic drawing of the wild type and truncated Sox2 fragments. (F) The interaction of Sox2 derivates with CARM1 in GST pull-down assays. GST or GST-Sox2 derivates were first incubated with whole cell extracts (WCEs) of HEK293T cells ectopically expressing FLAG-CARM1. The GST pull-down products were immunoblotted with anti-FLAG antibody.
    Streptavidin Conjugated Agarose Beads, supplied by Millipore, used in various techniques. Bioz Stars score: 92/100, based on 12 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/streptavidin conjugated agarose beads/product/Millipore
    Average 92 stars, based on 12 article reviews
    Price from $9.99 to $1999.99
    streptavidin conjugated agarose beads - by Bioz Stars, 2020-04
    92/100 stars
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    CARM1 is in association with Sox2. (A) Co-immunoprecipitation of Sox2 with CARM1. Whole cell extracts (WCEs) of MCF7 cells transfected with (+) or without (-) FLAG-CARM1 or GFP-Sox2, were subjected to immunoprecipitation with anti-FLAG antibody and blotted with antibodies against GFP or FLAG. (B) Co-precipitation of endogenous CARM1 with Sox2 by DNA pull-down. Biotin-labeled probes covering Sox2-binding sites in fgf4 and utf1 respectively were incubated with WCEs of P19 cells and streptavidin-conjugated agarose beads. The proteins precipitated with beads were denatured and resolved in SDS-PAGE and detected by immunoblotting with antibodies against Sox2 or CARM1. NC: negative control, no probe added. (C) Schematic drawing of the wild type and truncated CARM1 fragments. (D) GST pull-down assays to detect the interaction of Sox2 with CARM1 derivates. GST or GST-Sox2 was first incubated with WCEs of HEK293T cells ectopically expressing FLAG-CARM1 derivates. The GST pull-down products were immunoblotted with anti-FLAG antibody. Input: WCE control. (E) Schematic drawing of the wild type and truncated Sox2 fragments. (F) The interaction of Sox2 derivates with CARM1 in GST pull-down assays. GST or GST-Sox2 derivates were first incubated with whole cell extracts (WCEs) of HEK293T cells ectopically expressing FLAG-CARM1. The GST pull-down products were immunoblotted with anti-FLAG antibody.

    Journal: PLoS ONE

    Article Title: CARM1 Mediates Modulation of Sox2

    doi: 10.1371/journal.pone.0027026

    Figure Lengend Snippet: CARM1 is in association with Sox2. (A) Co-immunoprecipitation of Sox2 with CARM1. Whole cell extracts (WCEs) of MCF7 cells transfected with (+) or without (-) FLAG-CARM1 or GFP-Sox2, were subjected to immunoprecipitation with anti-FLAG antibody and blotted with antibodies against GFP or FLAG. (B) Co-precipitation of endogenous CARM1 with Sox2 by DNA pull-down. Biotin-labeled probes covering Sox2-binding sites in fgf4 and utf1 respectively were incubated with WCEs of P19 cells and streptavidin-conjugated agarose beads. The proteins precipitated with beads were denatured and resolved in SDS-PAGE and detected by immunoblotting with antibodies against Sox2 or CARM1. NC: negative control, no probe added. (C) Schematic drawing of the wild type and truncated CARM1 fragments. (D) GST pull-down assays to detect the interaction of Sox2 with CARM1 derivates. GST or GST-Sox2 was first incubated with WCEs of HEK293T cells ectopically expressing FLAG-CARM1 derivates. The GST pull-down products were immunoblotted with anti-FLAG antibody. Input: WCE control. (E) Schematic drawing of the wild type and truncated Sox2 fragments. (F) The interaction of Sox2 derivates with CARM1 in GST pull-down assays. GST or GST-Sox2 derivates were first incubated with whole cell extracts (WCEs) of HEK293T cells ectopically expressing FLAG-CARM1. The GST pull-down products were immunoblotted with anti-FLAG antibody.

    Article Snippet: In brief, 50 nmol 5′-end biotin-labeled oligo-DNA probes covering the Sox2-binding sites in fgf4 or utf1 were incubated with 1 mg whole cell extracts of P19 cells and streptavidin-conjugated agarose beads (Millipore) overnight at 4°C.

    Techniques: Immunoprecipitation, Transfection, Labeling, Binding Assay, Incubation, SDS Page, Negative Control, Expressing