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Immunofluorescence analysis of V5 tag was done on HEK-293 cells transiently overexpressing V5 - His-LacZ. The cells were fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. The cells were labeled with V5 Tag Mouse Monoclonal Antibody (R96025) at a dilution of 1:400 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Rabbit Anti-Mouse IgG Secondary Antibody (A11059) at a dilution of 1:400 for 30 minutes at room temperature (Panel a: green). Nuclei (Panel b: blue) were stained with SlowFade® Gold Antifade Mountant with DAPI (S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (A12381). Panel d is a merged image showing cellular localization. Panel e is untransfected HEK-293 cells. The images were captured using a Nikon microscope at 20X magnification.
|Tested species reactivity||Tag|
|Published species reactivity||Mouse, Human|
|Host / Isotype||Mouse / IgG2a|
|Immunogen||V5 synthetic peptide: Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr-.|
|Contains||0.01% sodium azide|
|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|
|Flow Cytometry (Flow)||1:100-1:500|
|Immunoprecipitation (IP)||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.
NOTE: R960-25 was previously sold under product #46-0705. When re-ordering, please use #R960-25.
R960-25 recognizes amino acid sequence: -Gly-Lys-Pro-Ile-Pro-Asn-Pro-Leu-Leu-Gly-Leu-Asp-Ser-Thr-.
This antibody is functionally tested against 20ng of an E. coli expressed fusion protein containing a V5 epitope using a chemiluminescent substrate at a 1 minute exposure. This antibody has also been tested in Western blot against 25ng 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.
This antibody has also been used successfully to immunoprecipitate fusion proteins that contain theV5 epitope.
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.
The FBXL10/KDM2B scaffolding protein associates with novel polycomb repressive complex-1 to regulate adipogenesis.
R960-25 was used in ChIP assay, immunoprecipitation, and western blot to examine the association of the FBXL10/KDM2B scaffolding protein with novel polycomb repressive complex-1 and its role in adipogenesis.
|Inagaki T,Iwasaki S,Matsumura Y,Kawamura T,Tanaka T,Abe Y,Yamasaki A,Tsurutani Y,Yoshida A,Chikaoka Y,Nakamura K,Magoori K,Nakaki R,Osborne TF,Fukami K,Aburatani H,Kodama T,Sakai J||The Journal of biological chemistry (290:4163)||2015|
Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.
R960-25 was used in western blot to study the role of human immunodeficiency virus type 1 enhancer-binding protein 3 in the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.
|Imamura K,Maeda S,Kawamura I,Matsuyama K,Shinohara N,Yahiro Y,Nagano S,Setoguchi T,Yokouchi M,Ishidou Y,Komiya S||The Journal of biological chemistry (289:9865)||2014|
New role of signal peptide peptidase to liberate C-terminal peptides for MHC class I presentation.
R960-25 was used in immunocytochemistry and western blot to study the involvement of signal peptide peptidase in MHC class I presentation.
|Oliveira CC,Querido B,Sluijter M,de Groot AF,van der Zee R,Rabelink MJ,Hoeben RC,Ossendorp F,van der Burg SH,van Hall T||Journal of immunology (Baltimore, Md. : 1950) (191:4020)||2013|
Critical differences between isoforms of securin reveal mechanisms of separase regulation.
R960-25 was used in western blot to characterize securin isoforms and their role in separase regulation.
|Han X,Poon RY||Molecular and cellular biology (33:3400)||2013|
In vivo cross-linking reveals principally oligomeric forms of α-synuclein and β-synuclein in neurons and non-neural cells.
R960-25 was used in western blot to study the existance of alpha- and beta-synuclein predomonantly as oligomers in normal neuronal and non-neuronal cells.
|Dettmer U,Newman AJ,Luth ES,Bartels T,Selkoe D||The Journal of biological chemistry (288:6371)||2013|
Cyclin E2 induces genomic instability by mechanisms distinct from cyclin E1.
R960-25 was used in western blot to investigate the effect of cyclin E1 and E2 on genomic instability.
|Caldon CE,Sergio CM,Burgess A,Deans AJ,Sutherland RL,Musgrove EA||Cell cycle (Georgetown, Tex.) (12:606)||2013|
Coordinate nuclear targeting of the FANCD2 and FANCI proteins via a FANCD2 nuclear localization signal.
R960-25 was used in immunocytochemistry and western blot to study the role of an amino terminal FANCD2 nuclear localization signal in the coordinated nuclear localization of both FANCD2 and FANCI.
|Boisvert RA,Rego MA,Azzinaro PA,Mauro M,Howlett NG||PloS one (8:null)||2013|
Epigenetic repression of cardiac progenitor gene expression by Ezh2 is required for postnatal cardiac homeostasis.
R960-25 was used in immunohistochemistry to study the role of Ezh2 during postnatal cardiac homeostasis.
|Delgado-Olguín P,Huang Y,Li X,Christodoulou D,Seidman CE,Seidman JG,Tarakhovsky A,Bruneau BG||Nature genetics (44:343)||2012|
46-0705; 460705; R960-25; R96025