anti flag conjugated beads sigma aldrich  (Roche)


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    Roche anti flag conjugated beads sigma aldrich
    MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), <t>immunoprecipitated</t> (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.
    Anti Flag Conjugated Beads Sigma Aldrich, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag conjugated beads sigma aldrich/product/Roche
    Average 97 stars, based on 1 article reviews
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    anti flag conjugated beads sigma aldrich - by Bioz Stars, 2021-07
    97/100 stars

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    1) Product Images from "Mitochondrial fission factor is a novel Myc-dependent regulator of mitochondrial permeability in cancer"

    Article Title: Mitochondrial fission factor is a novel Myc-dependent regulator of mitochondrial permeability in cancer

    Journal: EBioMedicine

    doi: 10.1016/j.ebiom.2019.09.017

    MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.
    Figure Legend Snippet: MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.

    Techniques Used: Permeability, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Flow Cytometry, Cytometry, Fluorescence, Incubation, Staining

    Related Articles

    Immunoprecipitation:

    Article Title: Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity
    Article Snippet: Cell lysate preparations, cell fractionations and western blot analysis were performed using standard protocols and are described in detail in the . .. Immunoprecipitation, mass spectrometry and identification of candidate interaction partners For CIC immunoprecipitation, myc-CIC-S or rabbit/mouse IgG (control) antibody-bound beads were prepared by incubating anti-CIC antibody, anti-myc antibody, normal rabbit-IgG, or mouse-IgG with protein G agarose beads (Roche) in IP buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA with 1X complete protease inhibitor from Roche) at 4°C overnight. .. Cell lysates were first pre-cleared with Sepharose 4B beads (Sigma) at 4°C for 1 hour and the lysates were immunoprecipitated with the prepared protein-G beads at 4ºC for 4 hours.

    Mass Spectrometry:

    Article Title: Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity
    Article Snippet: Cell lysate preparations, cell fractionations and western blot analysis were performed using standard protocols and are described in detail in the . .. Immunoprecipitation, mass spectrometry and identification of candidate interaction partners For CIC immunoprecipitation, myc-CIC-S or rabbit/mouse IgG (control) antibody-bound beads were prepared by incubating anti-CIC antibody, anti-myc antibody, normal rabbit-IgG, or mouse-IgG with protein G agarose beads (Roche) in IP buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA with 1X complete protease inhibitor from Roche) at 4°C overnight. .. Cell lysates were first pre-cleared with Sepharose 4B beads (Sigma) at 4°C for 1 hour and the lysates were immunoprecipitated with the prepared protein-G beads at 4ºC for 4 hours.

    Protease Inhibitor:

    Article Title: Mutations in CIC and IDH1 cooperatively regulate 2-hydroxyglutarate levels and cell clonogenicity
    Article Snippet: Cell lysate preparations, cell fractionations and western blot analysis were performed using standard protocols and are described in detail in the . .. Immunoprecipitation, mass spectrometry and identification of candidate interaction partners For CIC immunoprecipitation, myc-CIC-S or rabbit/mouse IgG (control) antibody-bound beads were prepared by incubating anti-CIC antibody, anti-myc antibody, normal rabbit-IgG, or mouse-IgG with protein G agarose beads (Roche) in IP buffer (20mM Tris-HCl pH 7.5, 150mM NaCl, 1mM EDTA with 1X complete protease inhibitor from Roche) at 4°C overnight. .. Cell lysates were first pre-cleared with Sepharose 4B beads (Sigma) at 4°C for 1 hour and the lysates were immunoprecipitated with the prepared protein-G beads at 4ºC for 4 hours.

    Article Title: Nuclear localization of the G protein ?5/R7-regulator of G protein signaling protein complex is dependent on R7 binding protein
    Article Snippet: Immunoprecipitation with anti-HA or anti-Xpress antibody was performed according to the manufacturer’s instructions (Immunoprecipitation Starter Pack, cat. No. 309410, Amersham Biosciences). .. Briefly, after transfection and 24 h incubation, PC12 cells were lysed with buffer A (250 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 0.5 % NP-40 and 1 X complete protease inhibitor cocktail [cat. No. 1836170, Roche]). .. The supernatant was incubated overnight with HA antibody at 4° C. The mixture was incubated with protein G-Sepharose previously equilibrated in buffer A for 2 hours at 4° C. The beads were then washed 3 times with buffer A by centrifugation at for 1 min, and the washes discarded.

    Article Title: Targeting CPT1A-mediated fatty acid oxidation sensitizes nasopharyngeal carcinoma to radiation therapy
    Article Snippet: CPT1 enzymatic activity was defined as millimoles of CoA-SH released per milligram of protein. .. Co-immunoprecipitation Cells were disrupted with IP lysis buffer containing protease inhibitor cocktails (Roche Diagnostics, Basel, Switzerland). .. Protein aliquots (500 μg) were pre-cleared by incubation with 20 μL of Dynabeads protein A (Invitrogen, MA, USA) for 1 h at 4 °C.

    Article Title: Parkin Mono-ubiquitinates Bcl-2 and Regulates Autophagy *
    Article Snippet: .. 293 cells co-expressing FLAG-parkin along with EGFP or EGFP-Bcl-2 were collected 48 h after transfection and sonicated in TSPI buffer containing 50 m m Tris-HCl, pH 7.5, 150 m m sodium chloride, 1 m m EDTA, 1% Nonidet P-40 supplemented with complete mini protease inhibitor mixture (Roche Applied Science). .. Cellular debris was removed by centrifugation at 12,000 × g for 30 min at 4 °C.

    Transfection:

    Article Title: KIF14 and citron kinase act together to promote efficient cytokinesis
    Article Snippet: Beads were washed twice with 1 ml lysis buffer and twice with 1 ml wash buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 40 mM β-glycerophosphate, 10 mM NaF, 0.1% [vol/vol] IGEPAL, and 1 mM Pefabloc). .. HEK293T cells plated on 15-cm–diameter dishes were transfected using 8 μg of the required plasmid DNA and 24 μl Fugene-6 (Roche Diagnostics) according to the manufacturer's instructions. .. After 40 h, cells were washed three times in ice-cold PBS and were lysed in immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% [vol/vol] IGEPAL, 2 mM Pefabloc, and complete protease inhibitor cocktail).

    Article Title: Nuclear localization of the G protein ?5/R7-regulator of G protein signaling protein complex is dependent on R7 binding protein
    Article Snippet: Immunoprecipitation with anti-HA or anti-Xpress antibody was performed according to the manufacturer’s instructions (Immunoprecipitation Starter Pack, cat. No. 309410, Amersham Biosciences). .. Briefly, after transfection and 24 h incubation, PC12 cells were lysed with buffer A (250 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 0.5 % NP-40 and 1 X complete protease inhibitor cocktail [cat. No. 1836170, Roche]). .. The supernatant was incubated overnight with HA antibody at 4° C. The mixture was incubated with protein G-Sepharose previously equilibrated in buffer A for 2 hours at 4° C. The beads were then washed 3 times with buffer A by centrifugation at for 1 min, and the washes discarded.

    Article Title: Parkin Mono-ubiquitinates Bcl-2 and Regulates Autophagy *
    Article Snippet: .. 293 cells co-expressing FLAG-parkin along with EGFP or EGFP-Bcl-2 were collected 48 h after transfection and sonicated in TSPI buffer containing 50 m m Tris-HCl, pH 7.5, 150 m m sodium chloride, 1 m m EDTA, 1% Nonidet P-40 supplemented with complete mini protease inhibitor mixture (Roche Applied Science). .. Cellular debris was removed by centrifugation at 12,000 × g for 30 min at 4 °C.

    Plasmid Preparation:

    Article Title: KIF14 and citron kinase act together to promote efficient cytokinesis
    Article Snippet: Beads were washed twice with 1 ml lysis buffer and twice with 1 ml wash buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 40 mM β-glycerophosphate, 10 mM NaF, 0.1% [vol/vol] IGEPAL, and 1 mM Pefabloc). .. HEK293T cells plated on 15-cm–diameter dishes were transfected using 8 μg of the required plasmid DNA and 24 μl Fugene-6 (Roche Diagnostics) according to the manufacturer's instructions. .. After 40 h, cells were washed three times in ice-cold PBS and were lysed in immunoprecipitation (IP) buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% [vol/vol] IGEPAL, 2 mM Pefabloc, and complete protease inhibitor cocktail).

    Article Title: A polyglutamine expansion disease protein sequesters PTIP to attenuate DNA repair and increase genomic instability
    Article Snippet: HEK293 cells with the PRS4-EGFP reporter gene were generated previously ( ). .. Stable HEK293 cells expressing full-length 3xFLAG-PTIP were generated by transfecting HEK293 cells with plasmid p3xFLAG-PTIP using Fugene 6 (Roche Molecular Biochemical). .. Stable clones were selected with 600 µg/ml of G418 for 10 days and then screened for PTIP expression through western blotting using an anti-flag antibody.

    Expressing:

    Article Title: A polyglutamine expansion disease protein sequesters PTIP to attenuate DNA repair and increase genomic instability
    Article Snippet: HEK293 cells with the PRS4-EGFP reporter gene were generated previously ( ). .. Stable HEK293 cells expressing full-length 3xFLAG-PTIP were generated by transfecting HEK293 cells with plasmid p3xFLAG-PTIP using Fugene 6 (Roche Molecular Biochemical). .. Stable clones were selected with 600 µg/ml of G418 for 10 days and then screened for PTIP expression through western blotting using an anti-flag antibody.

    Generated:

    Article Title: A polyglutamine expansion disease protein sequesters PTIP to attenuate DNA repair and increase genomic instability
    Article Snippet: HEK293 cells with the PRS4-EGFP reporter gene were generated previously ( ). .. Stable HEK293 cells expressing full-length 3xFLAG-PTIP were generated by transfecting HEK293 cells with plasmid p3xFLAG-PTIP using Fugene 6 (Roche Molecular Biochemical). .. Stable clones were selected with 600 µg/ml of G418 for 10 days and then screened for PTIP expression through western blotting using an anti-flag antibody.

    Incubation:

    Article Title: Nuclear localization of the G protein ?5/R7-regulator of G protein signaling protein complex is dependent on R7 binding protein
    Article Snippet: Immunoprecipitation with anti-HA or anti-Xpress antibody was performed according to the manufacturer’s instructions (Immunoprecipitation Starter Pack, cat. No. 309410, Amersham Biosciences). .. Briefly, after transfection and 24 h incubation, PC12 cells were lysed with buffer A (250 mM NaCl, 50 mM Tris, pH 8.0, 5 mM EDTA, 0.5 % NP-40 and 1 X complete protease inhibitor cocktail [cat. No. 1836170, Roche]). .. The supernatant was incubated overnight with HA antibody at 4° C. The mixture was incubated with protein G-Sepharose previously equilibrated in buffer A for 2 hours at 4° C. The beads were then washed 3 times with buffer A by centrifugation at for 1 min, and the washes discarded.

    Lysis:

    Article Title: Targeting CPT1A-mediated fatty acid oxidation sensitizes nasopharyngeal carcinoma to radiation therapy
    Article Snippet: CPT1 enzymatic activity was defined as millimoles of CoA-SH released per milligram of protein. .. Co-immunoprecipitation Cells were disrupted with IP lysis buffer containing protease inhibitor cocktails (Roche Diagnostics, Basel, Switzerland). .. Protein aliquots (500 μg) were pre-cleared by incubation with 20 μL of Dynabeads protein A (Invitrogen, MA, USA) for 1 h at 4 °C.

    Sonication:

    Article Title: Parkin Mono-ubiquitinates Bcl-2 and Regulates Autophagy *
    Article Snippet: .. 293 cells co-expressing FLAG-parkin along with EGFP or EGFP-Bcl-2 were collected 48 h after transfection and sonicated in TSPI buffer containing 50 m m Tris-HCl, pH 7.5, 150 m m sodium chloride, 1 m m EDTA, 1% Nonidet P-40 supplemented with complete mini protease inhibitor mixture (Roche Applied Science). .. Cellular debris was removed by centrifugation at 12,000 × g for 30 min at 4 °C.

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    Roche anti flag conjugated beads sigma aldrich
    MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), <t>immunoprecipitated</t> (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.
    Anti Flag Conjugated Beads Sigma Aldrich, supplied by Roche, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti flag conjugated beads sigma aldrich/product/Roche
    Average 97 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    anti flag conjugated beads sigma aldrich - by Bioz Stars, 2021-07
    97/100 stars
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    MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.

    Journal: EBioMedicine

    Article Title: Mitochondrial fission factor is a novel Myc-dependent regulator of mitochondrial permeability in cancer

    doi: 10.1016/j.ebiom.2019.09.017

    Figure Lengend Snippet: MFF regulation of mitochondrial outer membrane permeability. (a – c) PC3 cells were transfected with vector, Flag-MFF1 (a), Flag-MFF2 (b) or Flag-MFF5 (c), immunoprecipitated (IP) with an antibody to Flag and analysed by Western blotting. Sup, supernatant; Unb, unbound. (d) PC3 cells transfected with siCtrl or siMFF (top) or treated with H 2 O 2 (bottom) were labelled with calcein in the presence of CoCl 2 and analysed by flow cytometry. Numbers indicate fluorescence units per each condition. None, untreated. (e and f) PC3 cells transfected with siCtrl or siMFF were incubated with Fluoro3-AM and FLUO-5 N or, alternatively Rhod2-AM, and changes in mitochondrial (Mito)-, endoplasmic reticulum (ER)- or cytosolic (Cytosol)-associated Ca 2+ levels were determined by flow cytometry (e, representative experiment) and quantified (f). Numbers correspond to fluorescence units. Mean ± SD. (g) DU145 (top) or PC3 (bottom) cells were transfected with siCtrl or MFF-directed siRNA (siMFF) and analysed for mitochondrial inner membrane potential by TMRE staining and flow cytometry with or without suboptimal concentrations of the uncoupler, FCCP. Representative experiment ( n = 3). (h) The conditions are as in (g) and TMRE staining was quantified in siRNA-transfected DU145 and PC3 cells. Mean ± SD.

    Article Snippet: 2.6 Immunoprecipitation (IP) Cell extracts prepared in 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA containing 1% CHAPS, EDTA-free Protease Inhibitor Cocktail (Sigma-Aldrich) and Phosphatase Inhibitor Cocktail PhosSTOP (Roche) were immunoprecipitated with anti-Flag-conjugated beads (Sigma-Aldrich) and processed as described [ ].

    Techniques: Permeability, Transfection, Plasmid Preparation, Immunoprecipitation, Western Blot, Flow Cytometry, Cytometry, Fluorescence, Incubation, Staining