mouse monoclonal anti v5 hrp  (Thermo Fisher)


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    Name:
    V5 Mouse Monoclonal Antibody
    Description:
    V5 Mouse Monoclonal Antibody detects recombinant proteins containing the 14 amino acid V5 epitope The purified mouse Anti V5 antibody recognizes the sequence Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Antibody specifications• Applications western blotting immunofluorescence immunoprecipitation ELISA recommended dilution for western blot is 1 5 000 • Host species and isotype mouse IgG2aκ• Reactivity detects recombinant proteins containing the 14 amino acid V5 epitope
    Catalog Number:
    r960cus
    Price:
    None
    Applications:
    Antibodies and Secondary Detection|Cell Analysis|Cellular Imaging|Immunofluorescence (IF)|Immunofluorescence Staining & Detection|Immunoprecipitation|Protein Assays and Analysis|Protein Biology|Protein Purification & Isolation|Western Blot Detection|Western Blotting
    Category:
    Antibodies Secondary Detection Reagents
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    Structured Review

    Thermo Fisher mouse monoclonal anti v5 hrp
    Snk/Plk2 has kinase activity that is regulated by phosphorylation in its kinase domain. (a) Schematic of Snk/Plk2 mutants. Snk/Plk2 contains an N-terminal kinase domain and a C-terminal Polo box. Single-amino-acid residue substitutions in the kinase domain of conserved residues are indicated with an asterisk. Constructs contain a C-terminal V5-six-histidine tag. (b) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector. At 24 h, the cells were harvested for protein and analyzed for expression with mouse anti-V5 antibody. Actin was used to confirm that an equivalent amount of protein was loaded in each lane. (c) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector for 48 h. Equal amounts of 293 cell lysates (2.5 × 10 6 cell equivalent) were harvested and immunoprecipitated with mouse monoclonal anti-polyhistidine agarose and subjected to Western blot analysis (lower panel) or an in vitro protein kinase assay (upper panel) using casein as a substrate. Lysates were immunoblotted for exogenously immunoprecipitated Snk/Plk2 using mouse monoclonal <t>anti-V5-HRP.</t>
    V5 Mouse Monoclonal Antibody detects recombinant proteins containing the 14 amino acid V5 epitope The purified mouse Anti V5 antibody recognizes the sequence Gly Lys Pro Ile Pro Asn Pro Leu Leu Gly Leu Asp Ser Thr Antibody specifications• Applications western blotting immunofluorescence immunoprecipitation ELISA recommended dilution for western blot is 1 5 000 • Host species and isotype mouse IgG2aκ• Reactivity detects recombinant proteins containing the 14 amino acid V5 epitope
    https://www.bioz.com/result/mouse monoclonal anti v5 hrp/product/Thermo Fisher
    Average 99 stars, based on 1 article reviews
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    mouse monoclonal anti v5 hrp - by Bioz Stars, 2020-04
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    Images

    1) Product Images from "Silencing of the Novel p53 Target Gene Snk/Plk2 Leads to Mitotic Catastrophe in Paclitaxel (Taxol)-Exposed Cells"

    Article Title: Silencing of the Novel p53 Target Gene Snk/Plk2 Leads to Mitotic Catastrophe in Paclitaxel (Taxol)-Exposed Cells

    Journal: Molecular and Cellular Biology

    doi: 10.1128/MCB.23.16.5556-5571.2003

    Snk/Plk2 has kinase activity that is regulated by phosphorylation in its kinase domain. (a) Schematic of Snk/Plk2 mutants. Snk/Plk2 contains an N-terminal kinase domain and a C-terminal Polo box. Single-amino-acid residue substitutions in the kinase domain of conserved residues are indicated with an asterisk. Constructs contain a C-terminal V5-six-histidine tag. (b) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector. At 24 h, the cells were harvested for protein and analyzed for expression with mouse anti-V5 antibody. Actin was used to confirm that an equivalent amount of protein was loaded in each lane. (c) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector for 48 h. Equal amounts of 293 cell lysates (2.5 × 10 6 cell equivalent) were harvested and immunoprecipitated with mouse monoclonal anti-polyhistidine agarose and subjected to Western blot analysis (lower panel) or an in vitro protein kinase assay (upper panel) using casein as a substrate. Lysates were immunoblotted for exogenously immunoprecipitated Snk/Plk2 using mouse monoclonal anti-V5-HRP.
    Figure Legend Snippet: Snk/Plk2 has kinase activity that is regulated by phosphorylation in its kinase domain. (a) Schematic of Snk/Plk2 mutants. Snk/Plk2 contains an N-terminal kinase domain and a C-terminal Polo box. Single-amino-acid residue substitutions in the kinase domain of conserved residues are indicated with an asterisk. Constructs contain a C-terminal V5-six-histidine tag. (b) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector. At 24 h, the cells were harvested for protein and analyzed for expression with mouse anti-V5 antibody. Actin was used to confirm that an equivalent amount of protein was loaded in each lane. (c) 293 cells were transfected with the indicated Snk/Plk2 constructs or empty vector for 48 h. Equal amounts of 293 cell lysates (2.5 × 10 6 cell equivalent) were harvested and immunoprecipitated with mouse monoclonal anti-polyhistidine agarose and subjected to Western blot analysis (lower panel) or an in vitro protein kinase assay (upper panel) using casein as a substrate. Lysates were immunoblotted for exogenously immunoprecipitated Snk/Plk2 using mouse monoclonal anti-V5-HRP.

    Techniques Used: Activity Assay, Construct, Transfection, Plasmid Preparation, Expressing, Immunoprecipitation, Western Blot, In Vitro, Protein Kinase Assay

    Related Articles

    Clone Assay:

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: Individual clones were expanded and maintained in DMEM with hygromycin. .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera.

    Centrifugation:

    Article Title: Brain-Specific Regulator of G-Protein Signaling 9-2 Selectively Interacts with α-Actinin-2 to Regulate Calcium-Dependent Inactivation of NMDA Receptors
    Article Snippet: After centrifugation, immunoprecipitation pellets were washed five times using ice-cold wash buffer (50 m m Tris·Cl, pH 7.4, 5 m m EDTA, 300 m m NaCl, 0.1% Triton X-100, 0.02% sodium azide) with a brief centrifugation each time in a refrigerated microcentrifuge. .. Membranes were blocked and then incubated for 1 h at room temperature with mouse monoclonal anti-V5 antibody (1:5000; Invitrogen), goat anti-RGS9 antibody (directed against the RGS9 N terminus region; 1:300; T19; Santa Cruz Biotechnology), mouse anti-NR1 antibody (1:300; Chemicon), rabbit anti-α-actinin-2 (1:500), rabbit anti-DARPP-32 antibody (1:300; Santa Cruz Biotechnology), or mouse monoclonal anti-HA antibody (1:3000).

    Construct:

    Article Title: Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
    Article Snippet: .. Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C. .. Following incubation, cells were washed five times with 0.5% BSA in PBS and probed with an horseradish peroxidase–conjugated goat anti-mouse antibody (1:4000; 31430, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C.

    Article Title: Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism
    Article Snippet: The integrity of all constructs was confirmed by DNA sequence analysis. .. The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-β-tubulin (clone TUB 2.1), and rabbit polyclonal anti-βIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreticulin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen).

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: Full-length myc-tagged human tau (shortest four-repeat isoform, 4R0N) constructs (wild-type or P301L) and myc-tagged human CHIP were kindly provided by Dr. Leonard Petrucelli (Mayo Clinic, Jacksonville). .. The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    Enzyme-linked Immunosorbent Assay:

    Article Title: Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
    Article Snippet: Paragraph title: Cell ELISA ... Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C.

    Cell Surface Receptor Assay:

    Article Title: Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
    Article Snippet: For quantification of cell surface receptor expression, 5 × 104 HEK293 cells were plated in 96-well plates coated with 0.1 mg/ml poly- d -lysine. .. Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C.

    Incubation:

    Article Title: The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5
    Article Snippet: Immunocomplexes were incubated with protein G-agarose beads for 2 h at 4°C, washed three times with lysis buffer, resuspended in protein loading buffer, and resolved by polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose. .. Proteins were visualized by immunoblot analysis using horseradish peroxidase (HRP)-conjugated mouse anti-V5 mAb (Invitrogen), mouse anti-V5 mAb (Invitrogen) followed by anti-mouse HRP (Jackson), mouse anti-GFP mAb (Abcam) followed by anti-mouse HRP, or rat anti-B5 mAb followed by anti-rat HRP (Jackson) and developed with chemiluminescence reagents (Pierce) according to the manufacturer’s instructions.

    Article Title: Identification of direct targets and modified bases of RNA cytosine methyltransferases
    Article Snippet: .. Next the beads of each tube were re-suspended in 1.5ml of diluted modified RIPA buffer + BSA + 45μg of the Invitrogen’s mouse anti-V5 antibody (for replicate 1 & 2) or IgG (for control) and incubated at room temperature, rotating for 2 hours. .. The beads of each tube were then washed three times (2 minutes each) with 1ml of diluted modified RIPA buffer + BSA, and then re-suspended in 1.2 ml of diluted modified RIPA buffer + BSA + 15μl of RNasIN.

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *
    Article Snippet: Cells were incubated overnight at 4 °C with primary antibodies diluted in washing buffer containing 0.2% (v/v) Triton X-100 and 0.2% (w/v) BSA (immunoglobulin-free) in phosphate-buffered saline. .. The following antibodies were employed: mouse monoclonal anti-V5, working dilution 1:500 (Invitrogen, R96025); rabbit polyclonal anti-FLAG (Sigma, F7425; 1:500); mouse monoclonal anti-FLAG (Sigma, F3165; 1:5000); Alexa Fluor-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-mouse antibodies (1:4000; Life Technologies, Invitrogen, A-11008 and A-11005).

    Article Title: Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
    Article Snippet: .. Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C. .. Following incubation, cells were washed five times with 0.5% BSA in PBS and probed with an horseradish peroxidase–conjugated goat anti-mouse antibody (1:4000; 31430, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C.

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: For HA protein production, cells expressing either HA(1-330) or HA(1-500) were grown in T175 flask in serum-free medium and incubated for 24 hours. .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera.

    Article Title: Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase
    Article Snippet: .. Lysates were incubated with mouse anti-V5 antibody (Invitrogen) overnight at 4°, followed by addition of protein A/G-sepharose beads (Santa Cruz) for 1 hr at 4°. .. Beads were washed three times with wash buffer [50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA (pH 8.0)] supplemented with 1 μM ADP-HPD and phosphatase and protease inhibitor cocktail (1:100), and boiled with 4× sample buffer supplemented with 0.1 M DTT.

    Article Title: Brain-Specific Regulator of G-Protein Signaling 9-2 Selectively Interacts with α-Actinin-2 to Regulate Calcium-Dependent Inactivation of NMDA Receptors
    Article Snippet: .. Membranes were blocked and then incubated for 1 h at room temperature with mouse monoclonal anti-V5 antibody (1:5000; Invitrogen), goat anti-RGS9 antibody (directed against the RGS9 N terminus region; 1:300; T19; Santa Cruz Biotechnology), mouse anti-NR1 antibody (1:300; Chemicon), rabbit anti-α-actinin-2 (1:500), rabbit anti-DARPP-32 antibody (1:300; Santa Cruz Biotechnology), or mouse monoclonal anti-HA antibody (1:3000). .. Membranes were then incubated with HRP-conjugated secondary antibody (1:15,000 goat anti-mouse, goat anti-rabbit, or rabbit anti-goat; Sigma) for 1 h at room temperature.

    Introduce:

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: To generate HA-tagged mutant ubiquitin cDNAs, PCR-mediated site-directed mutagenesis was performed using the QuickChange XL kit (Stratagene) to introduce lysine (K) to arginine (R) substitutions at position 48 (K48R) or all lysine residues at positions 6, 11, 27, 29, 33, 48 and 63 (K0) through subsequent rounds of mutagenesis, as previously described ( ). .. The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    Expressing:

    Article Title: Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
    Article Snippet: For quantification of cell surface receptor expression, 5 × 104 HEK293 cells were plated in 96-well plates coated with 0.1 mg/ml poly- d -lysine. .. Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C.

    Article Title: Polymorphic variants of the human bile salt export pump (ABCB11): functional characterization and interindividual variability
    Article Snippet: .. The pEF6/V5-His-TOPO expression vector and monoclonal mouse Anti-V5 antibody were purchased from Invitrogen Corporation (Carlsbad, California, USA). ..

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera. .. Gel filtration chromatography Proteins at a concentration of 5 mg/ml were analyzed on Superdex S200 XK 16/60 column (GE Healthcare, Piscataway, NJ) pre-equilibrated with PBS, and the protein elution was monitored at 280 nm.

    Article Title: Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism
    Article Snippet: Paragraph title: Expression plasmids and antibodies ... The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-β-tubulin (clone TUB 2.1), and rabbit polyclonal anti-βIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreticulin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen).

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: Paragraph title: Expression plasmids, antibodies and recombinant proteins ... The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    Modification:

    Article Title: Identification of direct targets and modified bases of RNA cytosine methyltransferases
    Article Snippet: .. Next the beads of each tube were re-suspended in 1.5ml of diluted modified RIPA buffer + BSA + 45μg of the Invitrogen’s mouse anti-V5 antibody (for replicate 1 & 2) or IgG (for control) and incubated at room temperature, rotating for 2 hours. .. The beads of each tube were then washed three times (2 minutes each) with 1ml of diluted modified RIPA buffer + BSA, and then re-suspended in 1.2 ml of diluted modified RIPA buffer + BSA + 15μl of RNasIN.

    Western Blot:

    Article Title: Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers
    Article Snippet: The V5 epitope tag was detected using either mouse anti-V5 mAb (cat. R960-25, Thermo Fisher) used at 1:500 or a rabbit pAb (cat. AB3792, EMD Millipore) used at 1:300. .. Each of the primary antibodies was validated based on one or more methods, including prior use in multiple published studies with the same mAb or lot of polyclonal antiserum, manufacturer’s validation assays including knockouts, generation of expected immunofluorescence staining patterns, detection of appropriate band size on immunoblots without detection of non-specific bands, and detection of recombinant protein when expressed in cells that normally do not express the protein.

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera. .. Gel filtration chromatography Proteins at a concentration of 5 mg/ml were analyzed on Superdex S200 XK 16/60 column (GE Healthcare, Piscataway, NJ) pre-equilibrated with PBS, and the protein elution was monitored at 280 nm.

    Electron Microscopy:

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *
    Article Snippet: Immunofluorescence Microscopy 48 h after plating, tsA-201 cells were fixed in 4% (w/v) paraformaldehyde (Electron Microscopy Sciences, 15710) for 15 min at room temperature. .. The following antibodies were employed: mouse monoclonal anti-V5, working dilution 1:500 (Invitrogen, R96025); rabbit polyclonal anti-FLAG (Sigma, F7425; 1:500); mouse monoclonal anti-FLAG (Sigma, F3165; 1:5000); Alexa Fluor-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-mouse antibodies (1:4000; Life Technologies, Invitrogen, A-11008 and A-11005).

    Transfection:

    Article Title: Dishevelled enables casein kinase 1–mediated phosphorylation of Frizzled 6 required for cell membrane localization
    Article Snippet: .. Cells were transfected with 0.1 μg of the indicated constructs and maintained for an additional 24 h. Cells were then incubated with an anti-V5 antibody (mouse; 1:1000; R960-CUS, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C. .. Following incubation, cells were washed five times with 0.5% BSA in PBS and probed with an horseradish peroxidase–conjugated goat anti-mouse antibody (1:4000; 31430, Thermo Fisher Scientific) in 1% BSA in PBS for 1 h at 4 °C.

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: For control, mock transfected cells were all killed by hygromycin. .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera.

    Article Title: Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase
    Article Snippet: For immunoprecipitation experiments, S2R+ cells were harvested 48 hr after transfection, washed with 1× PBS, then lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA (pH 8.0)] supplemented with 1 μM ADP-HPD (Enzo Life Sciences) and phosphatase and protease inhibitor cocktail (1:100, Thermo Scientific). .. Lysates were incubated with mouse anti-V5 antibody (Invitrogen) overnight at 4°, followed by addition of protein A/G-sepharose beads (Santa Cruz) for 1 hr at 4°.

    Immunoprecipitation:

    Article Title: The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5
    Article Snippet: Paragraph title: Immunoprecipitation. ... Proteins were visualized by immunoblot analysis using horseradish peroxidase (HRP)-conjugated mouse anti-V5 mAb (Invitrogen), mouse anti-V5 mAb (Invitrogen) followed by anti-mouse HRP (Jackson), mouse anti-GFP mAb (Abcam) followed by anti-mouse HRP, or rat anti-B5 mAb followed by anti-rat HRP (Jackson) and developed with chemiluminescence reagents (Pierce) according to the manufacturer’s instructions.

    Article Title: Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase
    Article Snippet: Paragraph title: Immunoprecipitation ... Lysates were incubated with mouse anti-V5 antibody (Invitrogen) overnight at 4°, followed by addition of protein A/G-sepharose beads (Santa Cruz) for 1 hr at 4°.

    Article Title: Brain-Specific Regulator of G-Protein Signaling 9-2 Selectively Interacts with α-Actinin-2 to Regulate Calcium-Dependent Inactivation of NMDA Receptors
    Article Snippet: Paragraph title: Immunoprecipitation and immunoblotting. ... Membranes were blocked and then incubated for 1 h at room temperature with mouse monoclonal anti-V5 antibody (1:5000; Invitrogen), goat anti-RGS9 antibody (directed against the RGS9 N terminus region; 1:300; T19; Santa Cruz Biotechnology), mouse anti-NR1 antibody (1:300; Chemicon), rabbit anti-α-actinin-2 (1:500), rabbit anti-DARPP-32 antibody (1:300; Santa Cruz Biotechnology), or mouse monoclonal anti-HA antibody (1:3000).

    Protease Inhibitor:

    Article Title: Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase
    Article Snippet: For immunoprecipitation experiments, S2R+ cells were harvested 48 hr after transfection, washed with 1× PBS, then lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA (pH 8.0)] supplemented with 1 μM ADP-HPD (Enzo Life Sciences) and phosphatase and protease inhibitor cocktail (1:100, Thermo Scientific). .. Lysates were incubated with mouse anti-V5 antibody (Invitrogen) overnight at 4°, followed by addition of protein A/G-sepharose beads (Santa Cruz) for 1 hr at 4°.

    Infection:

    Article Title: The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5
    Article Snippet: For virus infections, HeLa cells were infected with the indicated viruses at an MOI of 5 and incubated overnight at 37°C. .. Proteins were visualized by immunoblot analysis using horseradish peroxidase (HRP)-conjugated mouse anti-V5 mAb (Invitrogen), mouse anti-V5 mAb (Invitrogen) followed by anti-mouse HRP (Jackson), mouse anti-GFP mAb (Abcam) followed by anti-mouse HRP, or rat anti-B5 mAb followed by anti-rat HRP (Jackson) and developed with chemiluminescence reagents (Pierce) according to the manufacturer’s instructions.

    Imaging:

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *
    Article Snippet: The following antibodies were employed: mouse monoclonal anti-V5, working dilution 1:500 (Invitrogen, R96025); rabbit polyclonal anti-FLAG (Sigma, F7425; 1:500); mouse monoclonal anti-FLAG (Sigma, F3165; 1:5000); Alexa Fluor-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-mouse antibodies (1:4000; Life Technologies, Invitrogen, A-11008 and A-11005). .. Images were captured with an Axiophot microscope (Carl Zeiss Inc., ×63, 1.4 numerical aperture Zeiss plan apochromat oil immersion lens) using a cooled CCD camera and at room temperature and Meta View image processing software (Universal Imaging Corp., West Chester, PA).

    Sequencing:

    Article Title: Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism
    Article Snippet: The integrity of all constructs was confirmed by DNA sequence analysis. .. The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-β-tubulin (clone TUB 2.1), and rabbit polyclonal anti-βIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreticulin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen).

    Affinity Purification:

    Article Title: Autoimmunity to ?IV spectrin in paraneoplastic lower motor neuron syndrome
    Article Snippet: The affinity-purified rabbit antiserum directed against a C-terminal peptide in βIVΣ1 spectrin (βIV-CT) has been described ( ). .. The following reagents were from commercial sources: mouse anti-V5 antibody (Invitrogen), anti-rabbit and anti-mouse goat IgG antibodies conjugated to Alexa 488 or Alexa 568 (Molecular Probes), rabbit IgG, peroxidase-conjugated goat anti-mouse, or goat anti-rabbit IgG and mouse anti-human IgG (Sigma).

    Recombinant:

    Article Title: Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers
    Article Snippet: The V5 epitope tag was detected using either mouse anti-V5 mAb (cat. R960-25, Thermo Fisher) used at 1:500 or a rabbit pAb (cat. AB3792, EMD Millipore) used at 1:300. .. Each of the primary antibodies was validated based on one or more methods, including prior use in multiple published studies with the same mAb or lot of polyclonal antiserum, manufacturer’s validation assays including knockouts, generation of expected immunofluorescence staining patterns, detection of appropriate band size on immunoblots without detection of non-specific bands, and detection of recombinant protein when expressed in cells that normally do not express the protein.

    Article Title: Polymorphic variants of the human bile salt export pump (ABCB11): functional characterization and interindividual variability
    Article Snippet: Recombinant vaccinia virus containing the T7-RNA polymerase gene (vtf-7) was a gift provided by Dr Bernard Moss (National Institutes of Health, Bethesda, Maryland, USA). .. The pEF6/V5-His-TOPO expression vector and monoclonal mouse Anti-V5 antibody were purchased from Invitrogen Corporation (Carlsbad, California, USA).

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: Paragraph title: Expression plasmids, antibodies and recombinant proteins ... The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    Immunofluorescence:

    Article Title: Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers
    Article Snippet: The V5 epitope tag was detected using either mouse anti-V5 mAb (cat. R960-25, Thermo Fisher) used at 1:500 or a rabbit pAb (cat. AB3792, EMD Millipore) used at 1:300. .. Each of the primary antibodies was validated based on one or more methods, including prior use in multiple published studies with the same mAb or lot of polyclonal antiserum, manufacturer’s validation assays including knockouts, generation of expected immunofluorescence staining patterns, detection of appropriate band size on immunoblots without detection of non-specific bands, and detection of recombinant protein when expressed in cells that normally do not express the protein.

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *
    Article Snippet: Paragraph title: Immunofluorescence Microscopy ... The following antibodies were employed: mouse monoclonal anti-V5, working dilution 1:500 (Invitrogen, R96025); rabbit polyclonal anti-FLAG (Sigma, F7425; 1:500); mouse monoclonal anti-FLAG (Sigma, F3165; 1:5000); Alexa Fluor-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-mouse antibodies (1:4000; Life Technologies, Invitrogen, A-11008 and A-11005).

    Nucleic Acid Electrophoresis:

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera. .. Gel filtration chromatography Proteins at a concentration of 5 mg/ml were analyzed on Superdex S200 XK 16/60 column (GE Healthcare, Piscataway, NJ) pre-equilibrated with PBS, and the protein elution was monitored at 280 nm.

    Fluorescence:

    Article Title: The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5
    Article Snippet: Proteins were visualized by immunoblot analysis using horseradish peroxidase (HRP)-conjugated mouse anti-V5 mAb (Invitrogen), mouse anti-V5 mAb (Invitrogen) followed by anti-mouse HRP (Jackson), mouse anti-GFP mAb (Abcam) followed by anti-mouse HRP, or rat anti-B5 mAb followed by anti-rat HRP (Jackson) and developed with chemiluminescence reagents (Pierce) according to the manufacturer’s instructions. .. Membranes were developed with chemiluminescence reagents (Pierce) according to the manufacturer’s directions, and both fluorescence and chemiluminescence signals were detected using a Kodak 4000MM Pro image station.

    Methylation:

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology). .. Recombinant human ubiquitin-carrier protein 7 (UbcH7) protein was obtained from Sigma; rabbit ubiquitin-activating enzyme E1 protein was obtained from Calbiochem; human Hsp70 protein was from Stressgen; human ubiquitin, K48R-ubiquitin and methylated-ubiquitin proteins were obtained from Affiniti Research; baculovirus-derived His6 -tagged human parkin protein has been described ( ).

    Mutagenesis:

    Article Title: Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism
    Article Snippet: Disease-associated or functional missense mutations (T12M, F182L, G504R, D513N, G533R, A467T and G788R) were introduced into V5-tagged WT ATP13A2 by site-directed mutagenesis using the Stratagene QuickChange II XL kit (Agilent Technologies, La Jolla, CA, USA) according to manufacturer's instructions. .. The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-β-tubulin (clone TUB 2.1), and rabbit polyclonal anti-βIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreticulin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen).

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: To generate HA-tagged mutant ubiquitin cDNAs, PCR-mediated site-directed mutagenesis was performed using the QuickChange XL kit (Stratagene) to introduce lysine (K) to arginine (R) substitutions at position 48 (K48R) or all lysine residues at positions 6, 11, 27, 29, 33, 48 and 63 (K0) through subsequent rounds of mutagenesis, as previously described ( ). .. The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    Microscopy:

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *
    Article Snippet: Paragraph title: Immunofluorescence Microscopy ... The following antibodies were employed: mouse monoclonal anti-V5, working dilution 1:500 (Invitrogen, R96025); rabbit polyclonal anti-FLAG (Sigma, F7425; 1:500); mouse monoclonal anti-FLAG (Sigma, F3165; 1:5000); Alexa Fluor-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-mouse antibodies (1:4000; Life Technologies, Invitrogen, A-11008 and A-11005).

    Purification:

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: Paragraph title: Protein expressing and purification ... The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera.

    Polymerase Chain Reaction:

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: To generate HA-tagged mutant ubiquitin cDNAs, PCR-mediated site-directed mutagenesis was performed using the QuickChange XL kit (Stratagene) to introduce lysine (K) to arginine (R) substitutions at position 48 (K48R) or all lysine residues at positions 6, 11, 27, 29, 33, 48 and 63 (K0) through subsequent rounds of mutagenesis, as previously described ( ). .. The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    Polyacrylamide Gel Electrophoresis:

    Article Title: The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5
    Article Snippet: Immunocomplexes were incubated with protein G-agarose beads for 2 h at 4°C, washed three times with lysis buffer, resuspended in protein loading buffer, and resolved by polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose. .. Proteins were visualized by immunoblot analysis using horseradish peroxidase (HRP)-conjugated mouse anti-V5 mAb (Invitrogen), mouse anti-V5 mAb (Invitrogen) followed by anti-mouse HRP (Jackson), mouse anti-GFP mAb (Abcam) followed by anti-mouse HRP, or rat anti-B5 mAb followed by anti-rat HRP (Jackson) and developed with chemiluminescence reagents (Pierce) according to the manufacturer’s instructions.

    Lysis:

    Article Title: The Ectodomain of the Vaccinia Virus Glycoprotein A34 Is Required for Cell Binding by Extracellular Virions and Contains a Large Region Capable of Interaction with Glycoprotein B5
    Article Snippet: Immunocomplexes were incubated with protein G-agarose beads for 2 h at 4°C, washed three times with lysis buffer, resuspended in protein loading buffer, and resolved by polyacrylamide gel electrophoresis (PAGE), followed by transfer to nitrocellulose. .. Proteins were visualized by immunoblot analysis using horseradish peroxidase (HRP)-conjugated mouse anti-V5 mAb (Invitrogen), mouse anti-V5 mAb (Invitrogen) followed by anti-mouse HRP (Jackson), mouse anti-GFP mAb (Abcam) followed by anti-mouse HRP, or rat anti-B5 mAb followed by anti-rat HRP (Jackson) and developed with chemiluminescence reagents (Pierce) according to the manufacturer’s instructions.

    Article Title: Wnt/Wingless Pathway Activation Is Promoted by a Critical Threshold of Axin Maintained by the Tumor Suppressor APC and the ADP-Ribose Polymerase Tankyrase
    Article Snippet: For immunoprecipitation experiments, S2R+ cells were harvested 48 hr after transfection, washed with 1× PBS, then lysed in lysis buffer [50 mM Tris-HCl (pH 8.0), 100 mM NaCl, 1% NP-40, 10% glycerol, 1.5 mM EDTA (pH 8.0)] supplemented with 1 μM ADP-HPD (Enzo Life Sciences) and phosphatase and protease inhibitor cocktail (1:100, Thermo Scientific). .. Lysates were incubated with mouse anti-V5 antibody (Invitrogen) overnight at 4°, followed by addition of protein A/G-sepharose beads (Santa Cruz) for 1 hr at 4°.

    Chromatin Immunoprecipitation:

    Article Title: Parkin mediates the degradation-independent ubiquitination of Hsp70
    Article Snippet: Full-length myc-tagged human tau (shortest four-repeat isoform, 4R0N) constructs (wild-type or P301L) and myc-tagged human CHIP were kindly provided by Dr. Leonard Petrucelli (Mayo Clinic, Jacksonville). .. The following antibodies were employed: mouse monoclonal antic-myc (clone 9E10), anti-c-myc-peroxidase, anti-HA (clone 12CA5) and anti-HA-peroxidase antibodies (Roche); mouse monoclonal anti-FLAG-(M2), anti-FLAG-(M2)-peroxidase, anti-β-tubulin and rabbit polyclonal anti-actin antibodies (Sigma); rabbit polyclonal anti-Hsp70 and mouse monoclonal anti-Hsp70 (clone SPA-810) antibodies (Stressgen); rabbit polyclonal anti-ubiquitin antibody (Dako); mouse monoclonal anti-V5 and anti-V5-peroxidase antibodies (Invitrogen); mouse monoclonal anti-tau (clone TAU-5) antibody (BD Biosciences); mouse monoclonal anti-polyubiquitinated conjugates (clone FK1) and anti-mono/polyubiquitinated conjugates (clone FK2) antibodies (Affiniti Research); mouse monoclonal anti-parkin (clone PRK8) antibody has been described ( ); rabbit polyclonal anti-superoxide dismutase 1 (SOD1) antibody was kindly provided by Dr. David Borchelt (University of Florida, Gainesville); peroxidase-coupled anti-mouse and anti-rabbit IgG secondary antibodies (Pierce Biotechnology).

    SDS Page:

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera. .. Gel filtration chromatography Proteins at a concentration of 5 mg/ml were analyzed on Superdex S200 XK 16/60 column (GE Healthcare, Piscataway, NJ) pre-equilibrated with PBS, and the protein elution was monitored at 280 nm.

    Plasmid Preparation:

    Article Title: Polymorphic variants of the human bile salt export pump (ABCB11): functional characterization and interindividual variability
    Article Snippet: .. The pEF6/V5-His-TOPO expression vector and monoclonal mouse Anti-V5 antibody were purchased from Invitrogen Corporation (Carlsbad, California, USA). ..

    Article Title: Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism
    Article Snippet: Expression plasmids containing human EGFP-LC3B (plasmid #11546; ), rat LAMP1-RFP (plasmid #1817, ), human RFP-Rab5A (plasmid #14437, ), human GFP-Rab7A (plasmid #12605; ), human GFP-Rab9A (plasmid #12663; ) and human GFP-Rab11A (plasmid #12674; ) were obtained from Addgene. .. The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-β-tubulin (clone TUB 2.1), and rabbit polyclonal anti-βIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreticulin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen).

    Software:

    Article Title: A Polybasic Plasma Membrane Binding Motif in the I-II Linker Stabilizes Voltage-gated CaV1.2 Calcium Channel Function *
    Article Snippet: The following antibodies were employed: mouse monoclonal anti-V5, working dilution 1:500 (Invitrogen, R96025); rabbit polyclonal anti-FLAG (Sigma, F7425; 1:500); mouse monoclonal anti-FLAG (Sigma, F3165; 1:5000); Alexa Fluor-488-conjugated goat anti-rabbit and Alexa-594-conjugated goat anti-mouse antibodies (1:4000; Life Technologies, Invitrogen, A-11008 and A-11005). .. Images were captured with an Axiophot microscope (Carl Zeiss Inc., ×63, 1.4 numerical aperture Zeiss plan apochromat oil immersion lens) using a cooled CCD camera and at room temperature and Meta View image processing software (Universal Imaging Corp., West Chester, PA).

    Functional Assay:

    Article Title: Common Pathogenic Effects of Missense Mutations in the P-Type ATPase ATP13A2 (PARK9) Associated with Early-Onset Parkinsonism
    Article Snippet: Disease-associated or functional missense mutations (T12M, F182L, G504R, D513N, G533R, A467T and G788R) were introduced into V5-tagged WT ATP13A2 by site-directed mutagenesis using the Stratagene QuickChange II XL kit (Agilent Technologies, La Jolla, CA, USA) according to manufacturer's instructions. .. The following antibodies were employed: mouse monoclonal anti-V5 and anti-V5-peroxidase (Invitrogen); mouse monoclonal anti-MAP2 (clone HM-2) and anti-β-tubulin (clone TUB 2.1), and rabbit polyclonal anti-βIII-tubulin (Sigma-Aldrich, Buchs, Switzerland); mouse monoclonal anti-HA-peroxidase (Roche Applied Science, Basel, Switzerland); mouse monoclonal anti-LAMP1 (clone LY1C6) and rabbit monoclonal anti-calreticulin (clone EPR3924) (EMD Millipore, Billerica, MA, USA); rabbit polyclonal anti-Giantin (ab24586; Abcam, Cambridge, UK); peroxidase-coupled anti-mouse and anti-rabbit IgG, light chain-specific secondary antibodies (Jackson ImmunoResearch, Inc., West Grove, PA, USA); anti-rabbit IgG and anti-mouse IgG coupled to AlexaFluor-488, -546 and -633 (Invitrogen).

    Affinity Chromatography:

    Article Title: A Rapid Flp-In System for Expression of Secreted H5N1 Influenza Hemagglutinin Vaccine Immunogen in Mammalian Cells
    Article Snippet: Then HA was purified from the cell supernatant by metal affinity chromatography using HisTrap columns (GE Healthcare, Piscataway, NJ). .. The expression of the HA proteins was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) under reducing and non-reducing conditions and western blotting using a mouse monoclonal anti-V5 antibody (Invitrogen), or a rabbit polyclonal anti-H5N1 HA1 specific sera.

    FLAG-tag:

    Article Title: Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers
    Article Snippet: The V5 epitope tag was detected using either mouse anti-V5 mAb (cat. R960-25, Thermo Fisher) used at 1:500 or a rabbit pAb (cat. AB3792, EMD Millipore) used at 1:300. .. The FLAG epitope tag was detected with anti-FLAG mAb M2 (cat. F1804, Sigma-Aldrich) used at 1:5000 dilution.

    Staining:

    Article Title: Efficient system for upstream mRNA trans-splicing to generate covalent, head-to-tail, protein multimers
    Article Snippet: The V5 epitope tag was detected using either mouse anti-V5 mAb (cat. R960-25, Thermo Fisher) used at 1:500 or a rabbit pAb (cat. AB3792, EMD Millipore) used at 1:300. .. Each of the primary antibodies was validated based on one or more methods, including prior use in multiple published studies with the same mAb or lot of polyclonal antiserum, manufacturer’s validation assays including knockouts, generation of expected immunofluorescence staining patterns, detection of appropriate band size on immunoblots without detection of non-specific bands, and detection of recombinant protein when expressed in cells that normally do not express the protein.

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    Thermo Fisher anti v5 hrp monoclonal antibody
    Anti V5 Hrp Monoclonal Antibody, supplied by Thermo Fisher, used in various techniques. Bioz Stars score: 99/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/anti v5 hrp monoclonal antibody/product/Thermo Fisher
    Average 99 stars, based on 3 article reviews
    Price from $9.99 to $1999.99
    anti v5 hrp monoclonal antibody - by Bioz Stars, 2020-04
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