V5 (mouse antibody (monoclonal)

Product no: AS21 4676

AS21 4676 | Clonality: Monoclonal  |  Host: Mouse  |  Reactivity: V5-tagged proteins

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  • Product Info
  • Sub class: IgG1a

    KLH-conjugated GKPIPNPLLGLDST synthetic peptide

    Host: Mouse
    Clonality: Monoclonal
    Purity: Affinity purified in PBS pH 7.4. Contains 0.02 % sodium azide. Contains 50 % glycerol.
    Format: Liquid
    Quantity: 50 µg
    Storage: Store at -20°C, Make aliquots to avoid repeated freeze-thaw cycles. Please remember to spin the tubes briefly prior to opening them to avoid any losses that might occur from material adhering to the cap or sides of the tube.
    Tested applications: ELISA (ELISA), Immunofluorescence (IF), Immunoprecipitation (IP), Western blot (WB)
    Recommended dilution: 1: 50 - 1: 200 (IF), 1: 200 - 1: 2000 (IP), 1: 500 -1 : 5000 (WB)
    Expected | apparent MW: Depends upon MW of fused protein
  • Reactivity
  • Confirmed reactivity: V5-tagged fusion proteins
  • Application Examples
  • Western blot using anti-V5 antibodies

    The antigen is a recombinant protein (GFP bright variant, called “Dasher)fused in C-tereminus to V5 epitope. Reagents and apparatus: SDS sample buffer (2x SDS, 0.1M DTT; 0.1M Tris-HCl, pH 8.0; 25mM EDTA; 0.01 % Bromophenol blue; 70% glycerol); SDS-PAGE 10× tricine running buffer (121gm Tris-Base, 179gm tricine, 10gm SDS, 900mL H20); Blotting buffer (48mM Tris, 39mM glycine, 1.3mM SDS, 200mL methanol diluted to 1L using H2O, pH not adjusted); TBS (0.05 M Tris, 0.9% NaCl adjust pH to 7.5); TBS-T (TBS plus 0.2% Tween 20).  Samples were boiled at 100 degree for 10 minutes in 1x final concentration of SDS sample buffer. Separated on a NuPage 4-12% SDS-PAGE  and blotted for 1 h to nitrocellulose membranes, using:  semi-dry transfer. Blot was blocked with 2% casein hydrolysate 4°C/ON. And 20 minutes at RT under agitation. Blot was incubated in the primary antibody at a dilution of 1:  10000 for 1h/RT with agitation in TBS-T with agitation. The antibody solution was decanted, and the blot was rinsed briefly twice, then washed once for 15 min and 3 times for 5 min in TBS-T at RT with agitation. Blot was incubated in  matching secondary antibody (anti-mouse IgG alkaline phosphatase conjugated) diluted to 1: 10000 in  for 1 h/RT with agitation. The blot was washed as above and developed with a chromogenic BCIP/NBT detection reagent. After 15 minutes, the blot was washed and the color development was stopped by decanting the staining solution and immersing the membranes into 20 mM EDTA solution.

    Courtesy of Dr.Giuseppe Dionisio,Aarhus University, Danmark

    Immunofluorescence using anti-V5 antibodies

    Immunofluorescencent analysis cells transfected with a V5-tag protein. Primary antibody at 1:2000 dilution (blue DAPI, red anti-V5).

  • Background
  • Background: V5-tag is a tag that can be added to a protein of interest as a fusion protein to enable purification and detection. It is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus (SV5). Addition of a V5-Tag to a protein of interest makes it possible to localize a specific gene product in a variety of cell types, study the topology of proteins and protein complexes, identify associated proteins, and characterize newly identified, low abundance or poorly immunogenic proteins when protein specific antibodies are not available.
  • Protocols
  • Agrisera Western Blot protocol and video tutorials

    Antibody Protocols
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