monoclonal anti flag m2  (Millipore)


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    Structured Review

    Millipore monoclonal anti flag m2
    BRCA2 minigenes and proteins expression. ( A ) Schematic representations of the BRCA2 minigenes. ( A.1 ) The wild-type full-length 11986bp-BRCA2 cDNA. ( A.2 ) The wild-type BRCA2 minigene encoding the C-terminal (CT) region of the BRCA2 protein. ( A.3 ) The mutagenized BRCA2 minigene carrying the c.8299C > T sequence variant encoding the p.Pro2767Ser variant DNA binding domain (DBD). ( A.4 ) The mutagenized BRCA2 minigene carrying the c.8297delC variant and encoding a truncated CT region lacking the DBD. Sequences encoding 3XFLAG were inserted at the N-terminal region. ( B ) A schematic representation of the cloned BRCA2-CT proteins. ( B.1 ) The cloned BRCA2-CT wt protein contains only the C-terminus domain of the BRCA2 protein. It ranges from aa 2634 to aa 3418, and contains part of the helical (H) domain (aa 2402–2668), three OB folds and the tower domain (T), the nuclear localization sequences (NLS), and the additional RAD51 binding domain (RAD51 BD). 3XFLAG is expressed at the N-terminal domain of this protein. ( B.2 ) BRCA2-CT P2767S contains the CT domain of the BRCA2 protein, as described above. The p.Pro2767Ser variant falls in the T domain. ( B.3 ) BRCA2-CT T2766NFs is a truncated protein derived from a premature stop codon insertion. It ranges from aa 2634 to aa 2765 and contains part of the H domain (aa 2402–2668) and only one of the three OB folds. It does not contain the NLS or any RAD51 binding domains. The c.8297delC variant falls in the OB1 fold. ( C ) A western blot analysis of the expression of BRCA2-CT wt, BRCA2-CT P2767S and BRCA2-CT T2766NFs proteins in NIH-3T3 cells. A mutant BRCA2 protein of 95 kDa was detected in cells that contained BRCA2-CT wt and BRCA2-CT P2767S proteins; a 15 kDa deleted BRCA2-CT T2766NFs protein derived from the Thr2766AsnFs mutation. ( D ) The nuclear localization of the BRCA2-CT wt, BRCA2-CT P2767S and the BRCA2-CT T2766NFs proteins in U2OS cells stained with the <t>anti-FLAG</t> antibody and analyzed by confocal microscopy. Scale bars 0.44 μm.
    Monoclonal Anti Flag M2, supplied by Millipore, used in various techniques. Bioz Stars score: 99/100, based on 53 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/monoclonal anti flag m2/product/Millipore
    Average 99 stars, based on 53 article reviews
    Price from $9.99 to $1999.99
    monoclonal anti flag m2 - by Bioz Stars, 2020-01
    99/100 stars

    Images

    1) Product Images from "A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair"

    Article Title: A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair

    Journal: Cancers

    doi: 10.3390/cancers11101454

    BRCA2 minigenes and proteins expression. ( A ) Schematic representations of the BRCA2 minigenes. ( A.1 ) The wild-type full-length 11986bp-BRCA2 cDNA. ( A.2 ) The wild-type BRCA2 minigene encoding the C-terminal (CT) region of the BRCA2 protein. ( A.3 ) The mutagenized BRCA2 minigene carrying the c.8299C > T sequence variant encoding the p.Pro2767Ser variant DNA binding domain (DBD). ( A.4 ) The mutagenized BRCA2 minigene carrying the c.8297delC variant and encoding a truncated CT region lacking the DBD. Sequences encoding 3XFLAG were inserted at the N-terminal region. ( B ) A schematic representation of the cloned BRCA2-CT proteins. ( B.1 ) The cloned BRCA2-CT wt protein contains only the C-terminus domain of the BRCA2 protein. It ranges from aa 2634 to aa 3418, and contains part of the helical (H) domain (aa 2402–2668), three OB folds and the tower domain (T), the nuclear localization sequences (NLS), and the additional RAD51 binding domain (RAD51 BD). 3XFLAG is expressed at the N-terminal domain of this protein. ( B.2 ) BRCA2-CT P2767S contains the CT domain of the BRCA2 protein, as described above. The p.Pro2767Ser variant falls in the T domain. ( B.3 ) BRCA2-CT T2766NFs is a truncated protein derived from a premature stop codon insertion. It ranges from aa 2634 to aa 2765 and contains part of the H domain (aa 2402–2668) and only one of the three OB folds. It does not contain the NLS or any RAD51 binding domains. The c.8297delC variant falls in the OB1 fold. ( C ) A western blot analysis of the expression of BRCA2-CT wt, BRCA2-CT P2767S and BRCA2-CT T2766NFs proteins in NIH-3T3 cells. A mutant BRCA2 protein of 95 kDa was detected in cells that contained BRCA2-CT wt and BRCA2-CT P2767S proteins; a 15 kDa deleted BRCA2-CT T2766NFs protein derived from the Thr2766AsnFs mutation. ( D ) The nuclear localization of the BRCA2-CT wt, BRCA2-CT P2767S and the BRCA2-CT T2766NFs proteins in U2OS cells stained with the anti-FLAG antibody and analyzed by confocal microscopy. Scale bars 0.44 μm.
    Figure Legend Snippet: BRCA2 minigenes and proteins expression. ( A ) Schematic representations of the BRCA2 minigenes. ( A.1 ) The wild-type full-length 11986bp-BRCA2 cDNA. ( A.2 ) The wild-type BRCA2 minigene encoding the C-terminal (CT) region of the BRCA2 protein. ( A.3 ) The mutagenized BRCA2 minigene carrying the c.8299C > T sequence variant encoding the p.Pro2767Ser variant DNA binding domain (DBD). ( A.4 ) The mutagenized BRCA2 minigene carrying the c.8297delC variant and encoding a truncated CT region lacking the DBD. Sequences encoding 3XFLAG were inserted at the N-terminal region. ( B ) A schematic representation of the cloned BRCA2-CT proteins. ( B.1 ) The cloned BRCA2-CT wt protein contains only the C-terminus domain of the BRCA2 protein. It ranges from aa 2634 to aa 3418, and contains part of the helical (H) domain (aa 2402–2668), three OB folds and the tower domain (T), the nuclear localization sequences (NLS), and the additional RAD51 binding domain (RAD51 BD). 3XFLAG is expressed at the N-terminal domain of this protein. ( B.2 ) BRCA2-CT P2767S contains the CT domain of the BRCA2 protein, as described above. The p.Pro2767Ser variant falls in the T domain. ( B.3 ) BRCA2-CT T2766NFs is a truncated protein derived from a premature stop codon insertion. It ranges from aa 2634 to aa 2765 and contains part of the H domain (aa 2402–2668) and only one of the three OB folds. It does not contain the NLS or any RAD51 binding domains. The c.8297delC variant falls in the OB1 fold. ( C ) A western blot analysis of the expression of BRCA2-CT wt, BRCA2-CT P2767S and BRCA2-CT T2766NFs proteins in NIH-3T3 cells. A mutant BRCA2 protein of 95 kDa was detected in cells that contained BRCA2-CT wt and BRCA2-CT P2767S proteins; a 15 kDa deleted BRCA2-CT T2766NFs protein derived from the Thr2766AsnFs mutation. ( D ) The nuclear localization of the BRCA2-CT wt, BRCA2-CT P2767S and the BRCA2-CT T2766NFs proteins in U2OS cells stained with the anti-FLAG antibody and analyzed by confocal microscopy. Scale bars 0.44 μm.

    Techniques Used: Expressing, Sequencing, Variant Assay, Binding Assay, Clone Assay, Derivative Assay, Western Blot, Mutagenesis, Staining, Confocal Microscopy

    2) Product Images from "Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling"

    Article Title: Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling

    Journal: Journal of Virology

    doi: 10.1128/JVI.02199-05

    Ebola virus VP35 binds to dsRNA. (A) 293T cells were transfected with 500 ng of the indicated protein expression plasmids (Luc, firefly luciferase). Twenty-four h posttransfection, cells were either mock infected (mock) or infected with SeV at an MOI of 8. Twelve h postinfection, cell lysates were prepared and incubated with pIC-Sepharose for 2 h at 4°C. Protein complexes were collected by centrifugation, washed 10 times, and separated by 12% sodium dodecyl sulfate-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane and visualized by Western blotting using anti-Flag M2 (WB: FLAG) or anti-VP35 6C5 (WB: VP35) monoclonal antibodies. To determine levels of protein expression, a fraction (2%) of the whole-cell extracts (WCE) was separated for Western blotting. (B) To assess the degree of impairment in dsRNA binding by the VP35 mutants, increasing amounts of lysates from cells expressing wild-type VP35 (VP35) or a VP35 mutant (R312A or K309A) were incubated with a fixed amount of pIC-Sepharose to obtain cell lysate/bead ratios of 0.05, 0.5, and 5 (wedges), and binding was assessed as in panel A. Input levels of wild-type and mutant VP35 proteins were assessed by Western blotting (WB: VP35). (C) The specificity for dsRNA binding was determined by competition assay. Soluble pIC, pAU, poly(rU) (pU), poly(rA) (pA), or dsDNA (from salmon sperm) at 12.5, 25, 50, and 100 μg/ml was added to cell lysates prior to addition of pIC-Sepharose (wedges, upper panel). Soluble dsRNA molecules were further tested at concentrations of 1, 10, 100, 1,000, and 10,000 ng/ml before pIC-Sepharose was added (wedges, lower panel). (D) Viral RNA was synthesized in vitro by using a T7-driven promoter cloned in front of VP35 sequences such that either positive-sense or negative-sense transcripts of different lengths were generated. Complementary ssRNAs were annealed in vitro to obtain the corresponding dsRNAs. These dsRNAs were analyzed on an agarose gel. Lanes: 1, 210 bp; 2, 409 bp; 3, 609 bp; 4, 809 bp; 5, 1,013 bp. A DNA ladder is present in the leftmost lane and contains molecules of the indicated sizes in base pairs. (E) Cell lysates containing wild-type VP35 were incubated without competitor (−) or with the indicated in vitro-transcribed dsRNA competitor molecules (see panel D) before pIC-Sepharose was added. The concentration of each dsRNA was 30 nM for each binding reaction. As a control, soluble pIC was used at 20 μg/ml. VP35 was detected by Western blotting as described previously. (F) The carboxy-terminal 171 amino acids of wild-type VP35 (C-171) were produced in a bacterial expression system with a C-terminal His tag and purified using a Talon Cobalt metal affinity column. Purified protein was used at ∼80 ng/ml in pIC-Sepharose binding assays as described previously, without (−) or with (+) soluble pIC as competitor. As a positive control, lysates from wild-type VP35-transfected 293T cells were run side by side. Input represents 2% of the total protein used for the immunoprecipitation. VP35 was detected with the C-terminal 10C7 monoclonal antibody.
    Figure Legend Snippet: Ebola virus VP35 binds to dsRNA. (A) 293T cells were transfected with 500 ng of the indicated protein expression plasmids (Luc, firefly luciferase). Twenty-four h posttransfection, cells were either mock infected (mock) or infected with SeV at an MOI of 8. Twelve h postinfection, cell lysates were prepared and incubated with pIC-Sepharose for 2 h at 4°C. Protein complexes were collected by centrifugation, washed 10 times, and separated by 12% sodium dodecyl sulfate-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane and visualized by Western blotting using anti-Flag M2 (WB: FLAG) or anti-VP35 6C5 (WB: VP35) monoclonal antibodies. To determine levels of protein expression, a fraction (2%) of the whole-cell extracts (WCE) was separated for Western blotting. (B) To assess the degree of impairment in dsRNA binding by the VP35 mutants, increasing amounts of lysates from cells expressing wild-type VP35 (VP35) or a VP35 mutant (R312A or K309A) were incubated with a fixed amount of pIC-Sepharose to obtain cell lysate/bead ratios of 0.05, 0.5, and 5 (wedges), and binding was assessed as in panel A. Input levels of wild-type and mutant VP35 proteins were assessed by Western blotting (WB: VP35). (C) The specificity for dsRNA binding was determined by competition assay. Soluble pIC, pAU, poly(rU) (pU), poly(rA) (pA), or dsDNA (from salmon sperm) at 12.5, 25, 50, and 100 μg/ml was added to cell lysates prior to addition of pIC-Sepharose (wedges, upper panel). Soluble dsRNA molecules were further tested at concentrations of 1, 10, 100, 1,000, and 10,000 ng/ml before pIC-Sepharose was added (wedges, lower panel). (D) Viral RNA was synthesized in vitro by using a T7-driven promoter cloned in front of VP35 sequences such that either positive-sense or negative-sense transcripts of different lengths were generated. Complementary ssRNAs were annealed in vitro to obtain the corresponding dsRNAs. These dsRNAs were analyzed on an agarose gel. Lanes: 1, 210 bp; 2, 409 bp; 3, 609 bp; 4, 809 bp; 5, 1,013 bp. A DNA ladder is present in the leftmost lane and contains molecules of the indicated sizes in base pairs. (E) Cell lysates containing wild-type VP35 were incubated without competitor (−) or with the indicated in vitro-transcribed dsRNA competitor molecules (see panel D) before pIC-Sepharose was added. The concentration of each dsRNA was 30 nM for each binding reaction. As a control, soluble pIC was used at 20 μg/ml. VP35 was detected by Western blotting as described previously. (F) The carboxy-terminal 171 amino acids of wild-type VP35 (C-171) were produced in a bacterial expression system with a C-terminal His tag and purified using a Talon Cobalt metal affinity column. Purified protein was used at ∼80 ng/ml in pIC-Sepharose binding assays as described previously, without (−) or with (+) soluble pIC as competitor. As a positive control, lysates from wild-type VP35-transfected 293T cells were run side by side. Input represents 2% of the total protein used for the immunoprecipitation. VP35 was detected with the C-terminal 10C7 monoclonal antibody.

    Techniques Used: Transfection, Expressing, Luciferase, Infection, Incubation, Centrifugation, Polyacrylamide Gel Electrophoresis, Western Blot, Binding Assay, Mutagenesis, Competitive Binding Assay, Synthesized, In Vitro, Clone Assay, Generated, Agarose Gel Electrophoresis, Concentration Assay, Produced, Purification, Affinity Column, Positive Control, Immunoprecipitation

    Related Articles

    Clone Assay:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Retrovirus Production and Infection —Full-length PTIP was cloned into pEF1A-HA-FLAG retroviral vector using the gateway system.

    Centrifugation:

    Article Title: Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice
    Article Snippet: Murine liver pieces were solubilized on ice in 1% NET-Triton buffer (150mM NaCl, 5mM EDTA, 10mM Tris, 1% Triton X-100, pH 7.4) with Complete Mini Protease Inhibitor Cocktail (Roche Diagnostic) and was cleared by centrifugation at 16 000 g for 30 minutes, and the supernatant was collected. .. Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International.

    Cotransfection:

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Transient expression of GBD fusion proteins in HEK293 cells after co-transfection was monitored by western-blot using an anti-Flag antibody (Sigma). .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Virus-containing supernatant was collected 48 and 72 h after co-transfection of pEF1A-HA-FLAG PTIP and pcl-ampho into BOSC23 packaging cells and were used to infect MEF cells in the presence of Polybrene.

    BIA-KA:

    Article Title: Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice
    Article Snippet: Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International. .. Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International.

    Construct:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: For coimmunoprecipitation assays in N. benthamiana , 3-week-old leaves were infiltrated with the indicated constructs and, after 3 d, treated with 50 μM for 24 h. Protein extracts were incubated with Anti-HA Affility Matrix (Roche) for 4 h at 4°C with rotation. .. For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ).

    Article Title: Model‐guided combinatorial optimization of complex synthetic gene networks
    Article Snippet: .. Antibodies Detection of FLAG‐tagged constructs was achieved using monoclonal ANTI‐FLAG M2 as primary antibody (Sigma, Cat # F1804) and an HRP‐linked secondary antibody (GE Healthcare, Cat # NA931). .. The signals were visualized with SuperSignalWest Femto MaxSubstrate (Life Technologies, Cat # 34095).

    Electrophoresis:

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: For immunoblotting, the agarose bead-protein complexes were separated by electrophoresis on 12.5% SDS-polyacrylamide gels, and the proteins were transferred to polyvinylidene difluoride membranes (Roche) by electrophoretic transfer. .. After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich).

    Incubation:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: For coimmunoprecipitation assays in N. benthamiana , 3-week-old leaves were infiltrated with the indicated constructs and, after 3 d, treated with 50 μM for 24 h. Protein extracts were incubated with Anti-HA Affility Matrix (Roche) for 4 h at 4°C with rotation. .. For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ).

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors ▿
    Article Snippet: After three washing steps (PBS), the cells were incubated with the respective primary antibody diluted in blocking buffer, followed by three washing steps and 30 min of incubation with anti-mouse Cy3-labeled Fab fragment (Amersham Biosciences, Little Chalfont, United Kingdom) for detection. .. Monoclonal antibody anti-Flag M2 (Sigma, St. Louis, MO) was used at a dilution of 1:1,000.

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature. .. Finally, after three washes with TBS-T, membranes were incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and images were captured using the LAS-3000 analyzer (Fujifilm).

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: .. After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich). .. After washing three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and visualized using Lumi-Light Western Blotting Substrate (Roche) in accordance with the manufacturer’s instructions.

    Recombinant:

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich). .. In brief, recombinant His-tagged MPK6 (10 μg) was activated by incubation with recombinant MKK5DD (1 μg) in the presence of 50 μ m ATP in 50 μL of reaction buffer (20 m m HEPES, pH 7.5, 10 m m MgCl2 , and 1 m m dithiothreitol) at 25°C for 30 min.

    Article Title: Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling
    Article Snippet: Monoclonal anti-FLAG (M2), poly(rA) · poly(rU) (pAU), poly(rU), and poly(rA) were from Sigma (Missouri). .. Recombinant human IFN-β was from Calbiochem (San Diego, CA).

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Antibodies —Rabbit polyclonal anti-PA1 antibodies were raised by immunizing rabbits with glutathione S -transferasefused PA1 recombinant protein. .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    Article Title: FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells
    Article Snippet: Antibodies and reagents Polyclonal antibodies against FIP200 or ULK1 were generated in rabbits by standard procedures with fragments of recombinant human FIP200 (residues 200–413 and 1–633) or mouse ULK1 (residues 738–1052) as antigens. .. Polyclonal anti-ULK1 antibody (A7481) and monoclonal anti-FLAG (M2) and anti–α-tubulin (DM1A) were purchased from Sigma-Aldrich.

    Article Title: De-ubiquitinating protease USP2a targets RIP1 and TRAF2 to mediate cell death by TNF
    Article Snippet: Human recombinant TNF and Fas and anti-caspase 8 antibody (C15) were obtained from Alexis Biochemicals (Enzo Life Sciences, Exeter, UK). .. Polyclonal and monoclonal anti-Haemagglutinin (HA), polyclonal anti-USP2a, polyclonal and monoclonal anti-Flag (M2), monoclonal β -actin, horseradish peroxydase-conjugated (HRP) goat-anti-rabbit were from Sigma-Aldrich (Dorset, UK).

    Cell Culture:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Two days later MEF cells were either irradiated as indicated or cultured in medium containing puromycin for the selection of stable clones.

    Expressing:

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Paragraph title: Detection of protein expression by western blot analysis ... Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Bradford Assay:

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Briefly, cells were lysed in 1X RIPA buffer (Sigma), containing 1% protease inhibitor cocktail set III, EDTA-free (Calbiochem), and protein concentration was determined by the Bradford assay. .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Western Blot:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: .. For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ). .. For immunodetection of 3HA-PYL8, anti-HA-HRP (Roche) was used at 1:1000 dilution.

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Paragraph title: Detection of protein expression by western blot analysis ... Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich). .. After washing three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and visualized using Lumi-Light Western Blotting Substrate (Roche) in accordance with the manufacturer’s instructions.

    Article Title: ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in Enterohemorrhagic Escherichia coli
    Article Snippet: .. Western blotting was performed with polyclonal anti-EspB ( ) or monoclonal anti-FLAG M2 (SIGMA) antibodies to probe EspB or FLAG tag, respectively. .. Binding of secondary anti-mouse immunoglobulin G antibody conjugated to horseradish peroxidase was detected using ECL Western blotting detection reagents (Amersham).

    Article Title: A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair
    Article Snippet: Paragraph title: 4.6. Protein Extracts and Western Blot Analysis ... The BRCA2 mutant proteins were immunodetected using the Monoclonal ANTI-FLAG® M2, Clone M2 (F1804, Sigma-Aldrich).

    Article Title: Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice
    Article Snippet: Paragraph title: Immunoblots ... Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International.

    Kinase Assay:

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: After washing three times with 20 m m Tris-HCl, pH 7.5, and 150 m m NaCl, the resultant agarose bead-protein complexes were used for either the kinase assay or immunoblotting. .. After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich).

    Transfection:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Transfections were performed using FuGENE 6 or Lipofectamine according to the manufacturer's instructions. .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    FLAG-tag:

    Article Title: ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in Enterohemorrhagic Escherichia coli
    Article Snippet: .. Western blotting was performed with polyclonal anti-EspB ( ) or monoclonal anti-FLAG M2 (SIGMA) antibodies to probe EspB or FLAG tag, respectively. .. Binding of secondary anti-mouse immunoglobulin G antibody conjugated to horseradish peroxidase was detected using ECL Western blotting detection reagents (Amersham).

    Protease Inhibitor:

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Briefly, cells were lysed in 1X RIPA buffer (Sigma), containing 1% protease inhibitor cocktail set III, EDTA-free (Calbiochem), and protein concentration was determined by the Bradford assay. .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Article Title: A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair
    Article Snippet: The nuclei pellet was suspended in 130 μL of nuclei lysis buffer (TRIS pH 7.5, 60 mM KCl, 1 mM DTT, 1 mM Na orthovanadate, 1 mM PMSF, 0.5 M Na fluoride and a protease inhibitor cocktail) and subjected to 3 cycles of freezing/thawing. .. The BRCA2 mutant proteins were immunodetected using the Monoclonal ANTI-FLAG® M2, Clone M2 (F1804, Sigma-Aldrich).

    Article Title: Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice
    Article Snippet: Murine liver pieces were solubilized on ice in 1% NET-Triton buffer (150mM NaCl, 5mM EDTA, 10mM Tris, 1% Triton X-100, pH 7.4) with Complete Mini Protease Inhibitor Cocktail (Roche Diagnostic) and was cleared by centrifugation at 16 000 g for 30 minutes, and the supernatant was collected. .. Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International.

    Infection:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Retrovirus Production and Infection —Full-length PTIP was cloned into pEF1A-HA-FLAG retroviral vector using the gateway system.

    Generated:

    Article Title: Ebola Virus VP35 Protein Binds Double-Stranded RNA and Inhibits Alpha/Beta Interferon Production Induced by RIG-I Signaling
    Article Snippet: Monoclonal anti-FLAG (M2), poly(rA) · poly(rU) (pAU), poly(rU), and poly(rA) were from Sigma (Missouri). .. Monoclonal anti-Ebola virus Zaire VP35 N-terminus (6C5) and C-terminus (17C6) antibodies were generated in collaboration with the Mount Sinai Hybridoma Center.

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: All deletion mutants were generated by site-directed mutagenesis (Stratagene) and verified by sequencing. .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    Article Title: FIP200, a ULK-interacting protein, is required for autophagosome formation in mammalian cells
    Article Snippet: Antibodies and reagents Polyclonal antibodies against FIP200 or ULK1 were generated in rabbits by standard procedures with fragments of recombinant human FIP200 (residues 200–413 and 1–633) or mouse ULK1 (residues 738–1052) as antigens. .. Polyclonal anti-ULK1 antibody (A7481) and monoclonal anti-FLAG (M2) and anti–α-tubulin (DM1A) were purchased from Sigma-Aldrich.

    Article Title: The Dual Effect of the Lupus-Associated Polymorphism rs10516487 on BANK1 gene Expression and Protein Localization
    Article Snippet: The polyclonal anti-human BANK1 (ET52) antibody was generated by immunization of rabbits with a synthetic peptide ETKHSPLEVGSESSC corresponding to amino acids 467 to 480 of the full-length BANK1 protein. .. Additional antibodies used in this study include the following: anti-V5 (Invitogen); monoclonal anti-β-tubulin, monoclonal anti-Flag M2 and rabbit anti-Flag (Sigma); anti-rabbit and anti-mouse IgG HRP (Zymed).

    Protein Concentration:

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Briefly, cells were lysed in 1X RIPA buffer (Sigma), containing 1% protease inhibitor cocktail set III, EDTA-free (Calbiochem), and protein concentration was determined by the Bradford assay. .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Sequencing:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: All deletion mutants were generated by site-directed mutagenesis (Stratagene) and verified by sequencing. .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    Affinity Purification:

    Article Title: TopBP1 Controls BLM Protein Level to Maintain Genome Stability
    Article Snippet: The monoclonal anti-FLAG M2, anti-HA, and anti–β-actin antibodies were purchased from Sigma-Aldrich. .. Anti-BLMpS338 polyclonal antibody was raised against phospho-peptide CKEDVLST-(phospho-S)-KDLLSKPE and affinity purified.

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: The antibody was affinity-purified using AminoLink Plus Immobilization and a purification kit (Pierce). .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    Article Title: The Dual Effect of the Lupus-Associated Polymorphism rs10516487 on BANK1 gene Expression and Protein Localization
    Article Snippet: The serum was further affinity purified against the peptides using the SulfoLink Kit (Pierce). .. Additional antibodies used in this study include the following: anti-V5 (Invitogen); monoclonal anti-β-tubulin, monoclonal anti-Flag M2 and rabbit anti-Flag (Sigma); anti-rabbit and anti-mouse IgG HRP (Zymed).

    Binding Assay:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors ▿
    Article Snippet: Monoclonal antibody anti-Flag M2 (Sigma, St. Louis, MO) was used at a dilution of 1:1,000. .. Immunofluorescence binding assays were done as described previously ( ).

    Article Title: ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in Enterohemorrhagic Escherichia coli
    Article Snippet: Western blotting was performed with polyclonal anti-EspB ( ) or monoclonal anti-FLAG M2 (SIGMA) antibodies to probe EspB or FLAG tag, respectively. .. Binding of secondary anti-mouse immunoglobulin G antibody conjugated to horseradish peroxidase was detected using ECL Western blotting detection reagents (Amersham).

    Immunofluorescence:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors ▿
    Article Snippet: Paragraph title: Immunofluorescence. ... Monoclonal antibody anti-Flag M2 (Sigma, St. Louis, MO) was used at a dilution of 1:1,000.

    Article Title: Acetylation of microtubules influences their sensitivity to severing by katanin in neurons and fibroblasts
    Article Snippet: Paragraph title: Immunofluorescence techniques ... Cy3 conjugated monoclonal anti-β-tubulin (1:150; for general tubulin staining), monoclonal anti-acetylated-tubulin (6-11B-1) (1:400), monoclonal anti-flag (M2) (1:500), and monoclonal anti-GFP (1:250; for the enhancement of GFP signals in experiments of neuron) antibodies were purchased from Sigma.

    Nucleic Acid Electrophoresis:

    Article Title: ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in Enterohemorrhagic Escherichia coli
    Article Snippet: Proteins in culture supernatants and whole-cell lysates from stationary-phase cultures, grown as above, were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting as described previously ( ). .. Western blotting was performed with polyclonal anti-EspB ( ) or monoclonal anti-FLAG M2 (SIGMA) antibodies to probe EspB or FLAG tag, respectively.

    Article Title: Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice
    Article Snippet: The liver tissue extracts (50 or 100 μg) were reduced and denatured with 3.6× Laemmli buffer for 5 minutes at 95°C and subjected to sodium dodecyl sulfate–polyacrylamide gel electrophoresis on 10% or 12% gels. .. Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International.

    Mutagenesis:

    Article Title: A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair
    Article Snippet: .. The BRCA2 mutant proteins were immunodetected using the Monoclonal ANTI-FLAG® M2, Clone M2 (F1804, Sigma-Aldrich). .. Actin, used to normalize the amount of protein in different samples, was revealed using an anti-β-actin mouse primary antibody (Sigma-Aldrich, 1:1000).

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: All deletion mutants were generated by site-directed mutagenesis (Stratagene) and verified by sequencing. .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    Immunodetection:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ). .. For immunodetection of 3HA-PYL8, anti-HA-HRP (Roche) was used at 1:1000 dilution.

    Labeling:

    Article Title: Model‐guided combinatorial optimization of complex synthetic gene networks
    Article Snippet: Antibodies Detection of FLAG‐tagged constructs was achieved using monoclonal ANTI‐FLAG M2 as primary antibody (Sigma, Cat # F1804) and an HRP‐linked secondary antibody (GE Healthcare, Cat # NA931). .. For tubulin Sigma′s T6199 monoclonal antibody was used and a fluorescently labeled secondary antibody visualized the signals (Li‐Cor, Cat # 92632210).

    Article Title: Hepatocyte-targeted HFE and TFR2 control hepcidin expression in mice
    Article Snippet: Immunoblot analysis was carried out with the use of monoclonal anti–FLAG M2 and mouse antiactin antibodies from Sigma-Aldrich, and rabbit anti-mTfR2 from Alpha Diagnostic International. .. Bands were detected by horseradish peroxidase–coupled secondary antibody and enhanced chemiluminescence (SuperSignal WestPico; Pierce Chemical) or by fluorescently labeled secondary antibodies as described previously.

    Purification:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ). .. The CDD complex was purified as described previously ( ).

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors ▿
    Article Snippet: Monoclonal antibody anti-Flag M2 (Sigma, St. Louis, MO) was used at a dilution of 1:1,000. .. Monoclonal antibody anti-myc 9E10 was purified from hybridoma supernatant by the same protocol as Fc fusion proteins.

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: The antibody was affinity-purified using AminoLink Plus Immobilization and a purification kit (Pierce). .. Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma.

    Protein Extraction:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: Paragraph title: Protein Extraction, Coimmunoprecipitation Assays, and Immunoblots ... For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ).

    Blocking Assay:

    Article Title: Kaposi's Sarcoma-Associated Herpesvirus gH/gL: Glycoprotein Export and Interaction with Cellular Receptors ▿
    Article Snippet: After three washing steps (PBS), the cells were incubated with the respective primary antibody diluted in blocking buffer, followed by three washing steps and 30 min of incubation with anti-mouse Cy3-labeled Fab fragment (Amersham Biosciences, Little Chalfont, United Kingdom) for detection. .. Monoclonal antibody anti-Flag M2 (Sigma, St. Louis, MO) was used at a dilution of 1:1,000.

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature. .. Finally, after three washes with TBS-T, membranes were incubated with ECL Prime Western Blotting Detection Reagent (GE Healthcare) and images were captured using the LAS-3000 analyzer (Fujifilm).

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: .. After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich). .. After washing three times, the membranes were incubated with horseradish peroxidase-conjugated secondary antibody and visualized using Lumi-Light Western Blotting Substrate (Roche) in accordance with the manufacturer’s instructions.

    Polyacrylamide Gel Electrophoresis:

    Article Title: ClpXP Protease Controls Expression of the Type III Protein Secretion System through Regulation of RpoS and GrlR Levels in Enterohemorrhagic Escherichia coli
    Article Snippet: Proteins in culture supernatants and whole-cell lysates from stationary-phase cultures, grown as above, were analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and Western blotting as described previously ( ). .. Western blotting was performed with polyclonal anti-EspB ( ) or monoclonal anti-FLAG M2 (SIGMA) antibodies to probe EspB or FLAG tag, respectively.

    Staining:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ). .. To confirm equal protein loading, membranes were stained with Ponceau reagent or immunoblotted using anti-RPT5 at 1:1000 dilution ( ).

    Article Title: Acetylation of microtubules influences their sensitivity to severing by katanin in neurons and fibroblasts
    Article Snippet: .. Cy3 conjugated monoclonal anti-β-tubulin (1:150; for general tubulin staining), monoclonal anti-acetylated-tubulin (6-11B-1) (1:400), monoclonal anti-flag (M2) (1:500), and monoclonal anti-GFP (1:250; for the enhancement of GFP signals in experiments of neuron) antibodies were purchased from Sigma. .. Rabbit polyclonal anti-GFP antibody (1:250; for the enhancement of GFP signals in experiment of ) was purchased (Abcam, Cambridge, UK).

    SDS Page:

    Article Title: Targeted Degradation of Abscisic Acid Receptors Is Mediated by the Ubiquitin Ligase Substrate Adaptor DDA1 in Arabidopsis [W]
    Article Snippet: After washing three times with 500 μL of extraction buffer, samples were separated on SDS-PAGE gels and transferred as described above. .. For immunoblots, membranes were probed with different antibodies: anti-GFP-HRP at 1:2000 dilution (for DDA1-GFP detection; Milteny Biotec), monoclonal anti-FLAG M2 at 1:1000 dilution (for FLAG-COP10; Sigma-Aldrich), anti-CUL4 at 1:500 dilution , anti-DET1 at 1:1000 dilution, and anti-DDB1 at 1:2000 dilution ( ).

    Article Title: Targeted DNA demethylation in human cells by fusion of a plant 5-methylcytosine DNA glycosylase to a sequence-specific DNA binding domain
    Article Snippet: Total cell lysates (80 μg) were subjected to SDS-PAGE (7% acrylamide/bisacrylamide, 37.5:1), and transferred to nitrocellulose membranes at 30 V at 4°C overnight. .. Then, membranes were incubated with Monoclonal Anti-Flag® M2, Clone M2 (F1804) antibody (Sigma) at a 1:1000 dilution in blocking solution at 4°C overnight, washed three times with TBS supplemented with 0.5% Tween-20 (TBS-T), and incubated with horseradish peroxidase-conjugated anti-mouse (Cell Signaling) at a 1:3000 dilution in blocking solution for 1 h at room temperature.

    Plasmid Preparation:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Retrovirus Production and Infection —Full-length PTIP was cloned into pEF1A-HA-FLAG retroviral vector using the gateway system.

    Software:

    Article Title: Model‐guided combinatorial optimization of complex synthetic gene networks
    Article Snippet: Antibodies Detection of FLAG‐tagged constructs was achieved using monoclonal ANTI‐FLAG M2 as primary antibody (Sigma, Cat # F1804) and an HRP‐linked secondary antibody (GE Healthcare, Cat # NA931). .. Quantification was done using ImageStudioLite software (Li‐Cor Biosciences).

    Irradiation:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Two days later MEF cells were either irradiated as indicated or cultured in medium containing puromycin for the selection of stable clones.

    Selection:

    Article Title: Accumulation of Pax2 Transactivation Domain Interaction Protein (PTIP) at Sites of DNA Breaks via RNF8-dependent Pathway Is Required for Cell Survival after DNA Damage
    Article Snippet: Both monoclonal anti-FLAG M2 and anti-β-actin antibodies were purchased from Sigma. .. Two days later MEF cells were either irradiated as indicated or cultured in medium containing puromycin for the selection of stable clones.

    Immunoprecipitation:

    Article Title: The MPK6-ERF6-ROS-Responsive cis-Acting Element7/GCC Box Complex Modulates Oxidative Gene Transcription and the Oxidative Response in Arabidopsis
    Article Snippet: Paragraph title: Immunoprecipitation, Immunoblotting, and Kinase Assays ... After blocking at room temperature, the membranes were incubated with monoclonal anti-FLAG M2, anti-AtMPK3, or anti-AtMPK6 antibody (Sigma-Aldrich).

    Lysis:

    Article Title: A Functional Analysis of the Unclassified Pro2767Ser BRCA2 Variant Reveals Its Potential Pathogenicity that Acts by Hampering DNA Binding and Homology-Mediated DNA Repair
    Article Snippet: The nuclei pellet was suspended in 130 μL of nuclei lysis buffer (TRIS pH 7.5, 60 mM KCl, 1 mM DTT, 1 mM Na orthovanadate, 1 mM PMSF, 0.5 M Na fluoride and a protease inhibitor cocktail) and subjected to 3 cycles of freezing/thawing. .. The BRCA2 mutant proteins were immunodetected using the Monoclonal ANTI-FLAG® M2, Clone M2 (F1804, Sigma-Aldrich).

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    F3165, supplied by Millipore, used in various techniques. Bioz Stars score: 90/100, based on 46 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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