Product Image Gallery
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 (Product # 37-7500) at a dilution of 1:500 in 1% BSA and incubated for 3 hours at room temperature and then labeled with Alexa Flour® 488 Rabbit Anti-Mouse IgG Secondary Antibody (Product # A-11059) 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 (Product # S36938). F-actin (Panel c: red) was stained with Alexa Fluor® 594 Phalloidin (Product # A12381). Panel d is a merged image showing cytoplasmic localization. Panel e is untransfected HEK-293 cells. The images were captured using a Nikon microscope at 20X magnification.
Western blot analysis of V5 tag was performed by loading 20 µg of whole cell extracts of untransfected HEK-293 and HEK-293 transiently overexpressing V5-His-LacZ using Novex® NuPAGE® 4-12% Bis-Tris gel (Product # NP0321BOX), XCell SureLock™ Electrophoresis System (Product # 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 Mouse V5 Tag Monoclonal Antibody (Product # 37-7500) at 1:500 dilution in 2.5% skim milk at 4°C overnight on a rocking platform. Goat Anti-Mouse IgG - HRP Secondary Antibody (Product # 62-6520) at 1:4000 dilution was used and chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (WP20005).
Published figure using V5 Tag monoclonal antibody (Product # 37-7500)
Figure 5. LAP2 depletion exacerbates the progerin-induced proliferation defect whereas specific overexpression of LAP2alpha rescues it. ( A ) Growth curve of normal (ctrl shRNA) and LAP2-depleted (shLAP2) primary fibroblasts expressing progerin or lamin A. Dotted lines indicate SEM (n = 3). Inset: growth rate after 8 days, error bras indicate SEM (*p < 0.05, Student's t -test). ( B ) Western blot of primary and TERT+ cells expressing v5-tagged lamin A (v5-LA) or progerin (v5-PG) with or without LAP2 silencing by shRNA (shLAP2). V5-tag, lamin A, lamin C, progerin (PG), LAP2alpha, LAP2beta and GAPDH are indicated. ( C ) Growth curve of fibroblasts expressing control vector or progerin in the presence or absence of doxycycline-inducible LAP2alpha. LAP2alpha expression was induced by addition of 0.25 ug/ml doxycycline. Dotted lines indicate SEM (n = 3). Inset: growth rate after 5 days, error bras indicate SEM (**p < 0.01, Student's t -test). ( D ) Western blot of primary fibroblasts carrying doxycycline-inducible v5-LAP2alpha, expressing control vector or progerin. LAP2alpha expression was induced by addition of 0.25 ug/ml doxycycline. LAP2alpha, progerin and actin are indicated. ( E ) Western blot showing doxycycline-dependent induction of v5-LAP2alpha in TERT+HGPS cells. ( F ) Box plot of H3K27me3 levels in human fibroblasts expressing progerin in the presence (red) or absence (blue) of ectopically expressed LAP2alpha (Student's t -test, p < 0.05, n > 7500 cell analyzed, whiske
Figure 2--figure supplement 1. ( A ) Western blot showing inducible expression of v5-progerin or v5-lamin A in primary mouse ESC +- doxycycline (DOX) as indicated. V5, nanog, GAPDH and actin are shown. ( B ) Immunofluorescence microscopy using v5-tag antibody (top panels) showing doxycycline-dependent expression of v5-lamin A and v5-progerin and localization to the nuclear periphery. ( C ) Immunofluorescence staining of embryoid body outgrowth. V5-tagged progerin (v5, green) and DNA damage foci (53BP-1, red) are shown. ( D ) Expression of v5-progerin in telomerase-deficient ESC. Western blot showing DOX-regulated expression of v5-progerin. Antibodies: v5-tag, lamin B1, GAPDH. ( E ) Immunofluorescence microscopy of Tert -/- ESC in the presence of absence of v5-progerin. Antibody: v5-tag (red), DAPI (blue). DOI: http://dx.doi.org/
Figure 1. Telomerase rescues dose dependent progerin-induced proliferation defects, DNA damage and gene expression changes without alleviating chromatin changes. ( A ) Immunofluorescence microscopy using v5-tag antibody showing doxycycline-dependent inducible expression of v5-progerin and its localization to the nuclear periphery. DAPI staining is shown on the bottom panels. Scale bar: 100 mum. ( B ) Western blotting showing doxycycline-dependent progerin expression in primary (left panel) and TERT+ (right panel) fibroblasts. Progerin migrates between lamin A and C as indicated (red arrowhead). Doxycycline concentrations (0-2000 ng/ml) are indicated under each lane. ( C ) Quantification of progerin-induced proliferation defects. Relative growth rates of primary (left panel) and TERT+ cells (right panel) according to progerin expression levels (*p < 0.05; ***p < 0.001 compared to control 0 ng/ml doxycycline, error bars represent SEM, 2-way ANOVA with Bonferroni's post-test). ( D ) Quantification of progerin-induced 53BP1 DNA damage foci in response to progerin expression levels, in primary (left panel, p < 0.01, chi 2 test) and TERT+ cells (right panel). 350-500 cells were counted for each condition. ( E ) Scatter plot analysis of primary (blue, red) and TERT+ (cyan, orange) cells showing an inverse correlation between H3K27me3 and progerin expression in each cell nucleus using immunofluorescence microscopy (Pearson r = -0.43 and -0.24 for TERT negative and TERT+ cells express
Figure 5--figure supplement 1. Depletion and expression of LAP2 in wild type and progerin expressing fibroblasts, control experiments. ( A ) Growth curve of primary and TERT+ control and LAP2 shRNA expressing fibroblasts. Dotted lines indicate SEM (n = 3). Inset: growth rate after 7 days. ( B ) Immunofluorescence microscopy analysis of lamin A/C and LAP2 levels in cells expressing scrambled (ctrl) or LAP2-specific shRNA (shLAP2). Scale bar: 20 mum. ( C ) Western blot of wild type cells expressing scrambled control shRNA (ctrl) or LAP2 shRNA (shLAP2). The LAP2 antibody recognizes both alpha and beta-isoforms of LAP2. Lamin A, lamin C and actin loading control are indicated. ( D ) Growth curve of normal (ctrl) and LAP2-depleted (shLAP2) TERT+ fibroblasts expressing progerin or lamin A. Dotted lines indicated SEM (n = 3). Inset: growth rate after 7 days. ( E ) Immunofluorescence microscopy showing dose-dependent induction of v5-LAP2alpha and its nucleoplasmic localization. Antibodies: v5, lamin B1 (LB1), merged + DAPI. Doxycyclin concentrations are indicated on the left. Scale bar: 50 mum. ( F ) Growth curve of primary fibroblasts expressing varying amounts of v5-LAP2alpha (doxycyclin concentration: 0, 0.25, 0.5 and 1 ug/ml). Dotted lines indicate SEM (n = 3). Inset: growth rate after 7 days (*p < 0.05, **p < 0.01, errors bars indicate SEM). ( G ) Western blot showing dose (doxycyclin)-dependent induction of v5-tagged LAP2alpha in normal dermal fibroblasts. Antibodies recognizin
Figure 3. BioID analysis reveals differential interaction of lamin A and progerin with lamina-associated polypeptide 2 (LAP2). ( A ) Western blot showing doxycycline-dependent expression of myc-BirA*-progerin (BirA-PG) and myc-BirA*-lamin A (BirA-LA) fusion constructs in primary and TERT+ cells. Antibodies are indicated: myc; lamin A, lamin C, LAP2alpha, actin, GAPDH. ( B ) Immunofluorescence microscopy confirms doxycycline-dependent induction and localization of BirA*-lamin A/BirA*-progerin fusion constructs to the nuclear periphery (green, myc tag; blue, DAPI staining). Scale bar: 20 mum. ( C ) Impaired interaction of LAP2 with progerin. Quantitative interactome of lamin A (black bars) or progerin (striped bars) with nuclear proteins lamin A, LAP2, lamin B1 and B2. Control: non-induced BirA*-lamin A (grey bars). BioID (emPAI) index: quantification based on the number of peptides for each protein detected by mass spectrometry error bars represent SEM (n = 3, ***p < 0.001, one-way ANOVA with Tukey's post-test). ( D ) Interaction of lamin A or progerin with LAP2alpha or emerin by co-immunoprecipitation. In vitro transcribed and translated v5-tagged lamin A, v5-tagged progerin, LAP2alpha and emerin (antibodies: v5-tag, LAP2alpha, emerin are indicated). Top panel: recombinant v5-tagged progerin and lamin A, myc-LAP2alpha and HA-emerin were efficiently immunoprecipitated using anti-v5-tag or anti-myc antibodies, respectively (input lanes two, three and four). Bottom panel: LAP2al
Figure 1--figure supplement 1. Progerin induced senescence, lamin B1 loss, DNA damage, and telomere shortening are prevented by TERT in primary and HGPS fibroblasts, control experiments. ( A ) Western blotting showing inducible expression of progerin or wild type lamin A in primary and TERT+ human fibroblasts +- doxycyclin (DOX) as indicated. ( B ) Immunofluorescence microscopy using V5-tag antibody (top panels) showing doxycyclin-dependent expression of v5-lamin A and v5-progerin in TERT+ human fibroblasts and localization to the nuclear periphery. Inset: higher magnification image of different field. DAPI staining is shown in bottom panels. Scale bar: 50 mum; scale bar inset: 20 mum. ( C ) Growth curve of TERT+ and primary cells in the presence or absence of progerin (+-DOX). Dotted lines indicate SEM (n = 5). Inset: growth rate after 6 days, error bars indicate SEM (**p < 0.01, one-way ANOVA with Tukey's post-test). ( D ) Quantification of lamin B1 levels upon progerin or lamin A expression in primary and TERT+ cells. Error bars indicate standard deviation (n = 4, *p = 0.05, Student's t -test). Values were normalized to no DOX control. ( E ) Quantification of progerin protein levels upon induction with doxycycline (+-DOX) in primary and TERT+ human fibroblasts. Levels were normalized to GAPDH loading control (n = 3, error bars indicate standard deviation). ( F ) Growth rate of primary and TERT+ fibroblasts in the presence (+DOX) or absence (-DOX) of exogenous lamin A (n =
Figure 6 Recombinant WDR73-V5 fusion protein rescues cell cycle defect in NCS patient fibroblasts. ( A and B ) Anti-V5 immunofluorescence (green) demonstrates that WDR73 C-terminal V5 fusion protein (WDR73-V5) overexpressed in NCS patient fibroblasts localizes to the mitotic microtubules (anti-alpha-tubulin) during metaphase ( A ) and telophase ( B ), rescuing the cell cycle defect in these cells. ( C-E ) Overexpression of WDR73 C-terminal V5 fusion protein (WDR73-V5) in HEK-293T cells. Anti-V5 immunofluorescence is in green; anti-alpha-tubulin in red. During pro-metaphase ( C ) and metaphase ( D ) recombinant WDR73-V5 colocalizes with alpha-tubulin at the mitotic spindle and aster microtubules. ( E ) WDR73-V5 localizes to the spindle poles, the kinetochore microtubules and the midzone microtubules during anaphase. Scale bars: A-E = 10 um. ( F-G ) Western blots of recombinant N-terminal FLAG WDR73 fusion proteins ( F ) and WDR73 C-terminal V5 fusion proteins ( G ) overexpressed for 40-48 h in HEK-293T cells. Anti-COX IV was labelled as a protein loading control. The abundance of FLAG-WDR73 p.Phe296Leufs*26 (F296Lfs*26) was 2.6- to 5.6-fold lower and FLAG-WDR73 p.Arg256Profs*18 (R256Pfs*18) was 1.6- to 6.5-fold lower than that of FLAG-WDR73 wild-type (wt) across four replicates despite transfection of equivalent amounts of plasmid DNA, suggesting instability of the truncated proteins.
Figure 4. Overexpression of LMNB1 causes senescence, which is reversible by telomerase or p53 inactivation. (a) Doxycyclin-inducible overexpression of v5-tagged LMNB1 (LB1). (top) Western blot showing LMNB1 levels upon doxycyclin induction (+) in hTERT-negative and hTERT-positive normal dermal fibroblasts. Samples were run in parallel on two gels and probed with the following antibodies: LMNB1, LMNA/C (LA and LC), and v5 tag. GAPDH loading control is shown for both gels. (bottom) Quantification of LMNB1 in noninduced versus induced cells (normalized to GAPDH/hTERT negative or hTERT positive/no doxycycline [Dox]; n = 5). The means +- SD of five independent experiments are shown. (b, top) Growth curve of hTERT-negative (red and light red) and hTERT-positive (blue and light blue) cells expressing LMNB1 or a vector control. Dotted lines indicate SEM ( n = 6). (bottom) Mean growth rate of cells +- LMNB1. Error bars indicate +- SEM. **, P < 0.01. (c) Quantification of SA-beta-gal-positive cells in LMNB1-overexpressing cells versus vector control ( n = 3). Data are presented as means +- SD from three independent experiments. (d) Expression of LMNB1 or vector control, dominant-negative p53 (p53DD), or pBABE-hygro control in hTERT-negative and hTERT-positive fibroblasts. Antibodies are p53, LMNB1, and GAPDH. (e) Growth curve of hTERT-deficient fibroblasts overexpressing LMNB1 (or pBABE-neo) in the presence of dominant-negative p53DD or pBABE-hygro control (ctrl). Dotted lines indicate
Published figure using V5 Tag monoclonal antibody (Product # 37-7500)
Western Blot (WB)
Host / Isotype
PBS, pH 7.4
0.1% sodium azide
Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.
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.
Have you cited this antibody in a publication?
so we can reference it in this datasheet.
Protein Aliases: V5 Epitope Tag
Suggested Secondary Antibodies
Material safety data sheets (MSDS)