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Antibodies

V5 Epitope Tag Antibody

Product Sizes
100 ug
600-401-378-100UG
About this Product
SKU:
600-401-378
Additional Names:
Rabbit Anti-V5 Epitope Tag Antibody, Rabbit Anti V5 Epitope Tag Antibody, Rabbit Anti-V5 Tag
Application:
ELISA, Immunofluorescence, Immunohistochemistry, Immunoprecipitation, Western Blot
Buffer:
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride
CE/IVD:
RUO
Clonality:
Polyclonal
Concentration:
1.8 mg/ml
Extra Details:
Epitope tags are short peptide sequences that are easily recognized by tag-specific antibodies. Due to their small size, epitope tags do not affect the tagged protein's biochemical properties. Most often sequences encoding the epitope tag are included with target DNA at the time of cloning to produce fusion proteins containing the epitope tag sequence. This allows anti-epitope tag antibodies to serve as universal detection reagents for any tag containing protein produced by recombinant means. This means that anti-epitope tag antibodies are a useful alternative to generating specific antibodies to identify, immunoprecipitate or immunoaffinity purify a recombinant protein. The anti-epitope tag antibody is usually functional in a variety of antibody-dependent experimental procedures. Expression vectors producing epitope tag fusion proteins are available for a variety of host expression systems including bacteria, yeast, insect and mammalian cells. Rockland Immunochemicals produces anti-epitope tag antibodies against many common epitope tags including Myc, GST, GFP, 6X His, MBP, FLAG, HA and V5. Rockland Immunochemicals also produces antibodies to other tags including FITC, Rhodamine (TRITC), DNP and biotin.
Formulation:
0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Host:
Rabbit
Immunogen:
This antibody was purified from whole rabbit serum prepared by repeated immunizations with V5 epitope tag peptide corresponding to aa 95-108 of the V protein conjugated to KLH using maleimide.
Isotype:
IgG
Physical State:
Liquid
Reactivities:
Label/Epitope-tag
Shipping Conditions:
Dry Ice
Specificity:
This affinity-purified antibody is directed against V5 motif and is useful in determining its presence in various assays. This polyclonal anti-V5-tag antibody detects over-expressed proteins containing the V5 epitope tag. To date this antibody has reacted with all V5 tagged proteins tested so far. In western blotting of bacterial extracts the antibody does not cross-react with endogenous proteins.
Storage Conditions:
-20[o]C aliquoted. Aliquot. Avoid freeze/thaw cycles.
Supplier:
Rockland Inc
Type:
Antibody: Tagged Antibodies
Anti-V5 epitope tag polyclonal antibody detects V5-tagged recombinant protein by western blot. This antibody was used at 1.0 ug/ml to detect 0.05 ug (lane 2) of full-length recombinant mouse serum albumin containing the V5 epitope tag at the carboxy end. Comparison to MW markers (lane 1) indicates detection of monomeric V5 tagged albumin. A 4-20% gradient gel was used to separate the protein by SDS-PAGE under non-reducing conditions. The protein was transferred to nitrocellulose using standard methods. After blocking the membrane was probed with the primary antibody overnight at 4° C followed by washes and reaction with a 1:10,000 dilution of IRDye 800 conjugated Gt-a-Rabbit IgG [H&L] (code 611-132-122) for 45 min at room temperature. LICOR's Odyssey® Infrared Imaging System was used to scan and process the image. Other detection systems will yield similar results.
Evi stabilization is dependent on Wnt palmitoylation. Schematic illustration of the Porcn-mediated Wnt palmitoylation, which is important for Evi-Wnt interaction and which is blocked upon Porcn inhibition (LGK974), in PorcnKO cells and by using a palmitoylation-deficient S209A Wnt3A mutant. Wild-type or stable Wnt3-and Wnt5B-expressing HEK293T cells were treated with 5 uM LGK974 for 48 h and subjected to Western blot analysis. Western blot analysis of endogenous Evi in wt, PorcnKO, or EviKO HEK293T cells upon overexpression of Wnt3A or IGFBP5-V5. PorcnKO1.2 and PorcnKO1.4 indicate clone #2 and clone #4 of PorcnKO HEK293T cells generated with Porcn sgRNA1 (Appendix Fig S3). Clonal EviKO HEK293T cells were generated with Evi sgRNA2 (EviKO2.9; clone #9) or Evi sgRNA1 (EviKO1.1; clone #1; Appendix Fig S2). Increase in total B Beta-catenin protein served as control for Wnt pathway activation. Western blot analysis of endogenous Evi in HEK293T cells transfected with the indicated overexpression plasmids. When indicated, the cells were additionally treated with 5 uM LGK974 for 48 h. Western blot analysis of endogenous Evi in HCT116 or A375 cells treated with 5 uM LGK974 or DMSO for the indicated hours (h). All Western blots are representative of three independent experiments. B Beta-Actin was used as a loading control, LRP6 as a reference membrane protein and EGF-Myc and IGFBP5-V5 as controls for secreted proteins. Specific Evi bands are indicated by arrows, and unspecific bands are marked by asterisks. Scheme: Wnt-induced Evi stabilization is blocked in the absence of Wnt palmitoylation (PorcnKO, LGK974, Wnt3A S209A).Source data are available online for this figure. Figure provided by CiteAb. Source: EMBO J, PMID: 29378775.
Evi is not transcriptionally regulated by WntA, BFPKM-normalized RNA-seq. data of the TCGA Research Network (TCGA-COAD; http://cancergenome.nih.gov/; 09/25/2017) were log-transformed to illustrate the relative expression of (A) Wnt3 and (B) Evi in healthy colon (41) versus colon adenocarcinoma (456). The distribution into tumor and healthy samples was based on their barcodes as described in TCGA Wiki. Statistical significance of gene expression differences was determined using a Student's t-test under the alternative hypothesis H1 that gene expression is higher in tumors compared to healthy tissue. The boxplot diagram shows the median as line within the box, the 25th and 75th percentiles as the upper and lower part of the box, the 10th and 90th percentiles as error bars and outliers as circles.CHEK293T cells were transfected with the indicated expression constructs, treated with 100 ng/ml recombinant mouse Wnt3A (rec. W3A) or with 10 uM GSK3 inhibitor SB216763 for the indicated hours (h). AXIN2 and Evi mRNA levels were analyzed by qRT-PCR and normalized to GAPDH expression. Results are shown as mean +/- s.d. from three independent experiments. D, D'Twenty-four hours after reverse transfection with Ctrl or CTNNB1 siRNA, HEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression plasmids and analyzed (D) for the indicated proteins via immunoblotting or (D') for canonical Wnt activity using the TCF-Luciferase Wnt reporter assay. Immunoblotting is representative of three independent experiments, and Wnt reporter activity was calculated as mean from three independent experiments +/- s.d.Source data are available online for this figure. Figure provided by CiteAb. Source: EMBO J, PMID: 29378775.
Evi poly-ubiquitination is regulated by the presence of Wnt proteinsHEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression constructs and treated with MG132 at the indicated concentrations for 24 h. Cell lysates were analyzed for endogenous Evi by immunoblotting. Total B Beta-catenin protein was used to assess MG132 efficiency. Wild-type (wt), stable Wnt3-expressing, or EviKO2.9 HEK293T cells were treated with 1 uM MG132 for 24 h. TUBE2 immunoprecipitates were assayed for endogenous Evi or K48 poly-ubiquitin by immunoblotting. To confirm specificity of the TUBE2 assay, Ctrl agarose beads were used as control. The asterisk at the B Beta-actin blot indicates Wnt3A proteins blotted before membrane stripping. Scheme illustrating ubiquitination and proteasomal degradation of Evi, which is blocked in the presence of Wnt ligands.HEK293T cells were transfected with the indicated plasmids and additionally treated with 5 uM LGK974 for 48 h when indicated. In case of Wnt3A-KDEL, the ER-retaining sequence KDEL was C-terminally fused to Wnt3A. Dvl2-HA, Dvl3, and B Beta-catenin-YFP overexpression was used as negative control to verify that Evi stabilization was not due to downstream activation of Wnt signaling. All Western blots are representative of three independent experiments. B Beta-Actin was used as loading control. Specific Evi bands are marked by arrows and unspecific bands by asterisks. Source data are available online for this figure. Figure provided by CiteAb. Source: EMBO J, PMID: 29378775.
Wnt ligand production increases Evi protein levels In situ RNA hybridization (red dots) and immunohistochemistry (brown staining) of Evi were performed on sequential FFPE tissue slides of healthy colon and matched colon cancer tissue from five patients. The illustrated example is representative for three patients. Scale bar: 40 um. Specificity of Evi probes was confirmed in Appendix Fig S1B.Wild-type (wt) or EviKO HEK293T cells were transfected with Wnt3A or IGFBP5-V5 expression plasmids and subjected to Western blot analysis. Specific Evi bands are indicated by arrows and unspecific bands by asterisks. Endogenous Evi is not only detectable as a monomeric form (46 kDa) but also as SDS-resistant dimers (80 kDa). Clonal EviKO HEK293T cells were generated via CRISPR/Cas9 using Evi sgRNA #2 (EviKO2.9) or Evi sgRNA #1 (EviKO1.1; Appendix Fig S2).HEK293T cells were transfected with Wnt expression plasmids and analyzed for endogenous Evi levels by immunoblotting with a mouse monoclonal Evi antibody (Biolegend, clone YJ5).HEK293T cells were transfected with the indicated overexpression constructs, treated with 100 ng/ml recombinant mouse Wnt3A (rec. W3A) or with 10 uM GSK3B Beta inhibitor SB216763 for the indicated hours (h). The obtained cell lysates were used for Western blot analysis. Representative Western blots of three independent experiments are shown. B Beta-Actin or N-cadherin were used as a loading control, and LRP6 served as a reference membrane protein. Scheme showing that Evi is regulated through Wnt proteins within the Wnt-producing cell. Canonical Wnt signaling can be activated by Wnt ligands, Dishevelled (Dvl) overexpression or by the GSK3B Beta inhibitor SB216763.Source data are available online for this figure. Figure provided by CiteAb. Source: EMBO J, PMID: 29378775.