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Western blot analysis of V5 tag was performed by loading 20 µg whole cell extracts of untransfected HEK-293 and HEK-293 transiently overexpressing V5-His-LacZ using Novex® NuPAGE® 4-12% Bis-Tris gel (NP0321BOX), XCell SureLock™ Electrophoresis System (EI0002), and Novex® Sharp Pre-Stained Protein Standard (LC5800). Proteins were transferred to a PVDF membrane and blocked with 5% skim milk for 1 hour at room temperature. V5-His-LacZ was detected at ~117 kDa using V5-HRP Mouse Monoclonal Antibody (R96125) at 1:3000 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
|Tested species reactivity||Tag|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||V5 synthetic peptide: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr-.|
|Storage Conditions||Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.|
|Tested Applications||Dilution *|
|ELISA (ELISA)||Assay dependent|
|Western Blot (WB)||1:1000-1:5000|
* Suggested working dilutions are given as a guide only. It is recommended that the user titrate the product for use in their own experiment using appropriate negative and positive controls.
R961-25 recognizes amino acid sequence: -Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr-.
This antibody is functionally tested against 500ng of an E. coli expressed fusion protein containing a V5 epitope using a chemiluminescent substrate at a 10 minute exposure. This antibody has also been tested in Western blot against 50ng of recombinant Positope™ protein. The Positope™ control protein is a 53 kDa recombinant protein that contains seven epitope tags, including His (C-term), HisG, c-myc, and V5. Low background was observed using chemiluminescent or alkaline phosphatase reagents for detection. For Western blot, dilute in PBS or Tris-Buffered Saline (TBS) containing 0.05% Tween-20 and 5% nonfat, dry milk (PBSTM or TBSTM).
Using chemiluminescence as the detection method, no cross-reactivity has been observed in bacterial lysates. In mammalian lysates, a few cross-reactive proteins have been observed upon overexposure of blots.
Epitope tags provide a method to localize gene products 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. The V5 epitope tag is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus (SV5). The V5 tag is usually used with all 14 amino acids (GKPIPNPLLGLDST), although it has also been used with a shorter 9 amino acid sequence (IPNPLLGLD).
For Research Use Only. Not for use in diagnostic procedures. Not for resale without express authorization.