V5 Epitope Tag Antibody, Unconjugated, Rabbit, Polyclonal Preis auf Anfrage
Biozol Catalog Number:
BYT-ORB345390
Supplier Catalog Number:
orb345390
Alternative Catalog Number:
BYT-ORB345390-100
Manufacturer:
Biorbyt
Host:
Rabbit
Category:
Antikörper
Application:
ELISA, IHC, WB
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.
Conjugation:
Unconjugated
Alternative Names:
Rabbit Anti-V5 Epitope Tag Antibody, Rabbit Anti V5 Epitope Tag Antibody, Rabbit Anti-V5 Tag
Preservative: 0.01% (w/v) Sodium Azide. Stabilizer: None, Buffer: 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Purity:
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. The antibody recognizes the V5-epitope tag (GKPIPNPLLGLDST) fused to either the carboxy- terminal end of targeted proteins in transfected or transformed cells. Although not yet tested, expect reactivity with recombinant proteins prepared with the V5-epitope tag fused to the amino terminal end as well.
Application Notes: Anti-V5 is optimally suited for monitoring expression of V5-tagged fusion proteins. The V5 epitope tag is derived from a small epitope (Pk) present on the P and V proteins of the paramyxovirus of simian virus 5 (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). This antibody has been tested by ELISA and western blotting against both the immunizing peptide and V5 containing recombinant proteins. Although not tested, this antibody is likely functional for immunoprecipitation and immunocytochemistry
Anti-V5 epitope tag polyclonal antibody detects V5-tagged recombinant protein by western blot. This antibody was used at 1.0 µg/ml to detect 0.05 µg (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 4C followed by washes and reaction with a 1:10000 dilution of IRDye 800 conjugated Gt-a-Rabbit IgG [H&L] for 45 min at room temperature.
Evi is not transcriptionally regulated by WntA, BFPKM-normalized RNA-seq. data of the TCGA Research Network 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 Students 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 µm 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, DTwenty-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.
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 beta-catenin protein was used to assess MG132 efficiency. Wild-type (wt), stable Wnt3-expressing, or EviKO2.9 HEK293T cells were treated with 1 µm 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 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 µm 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 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. beta-Actin was used as loading control. Specific Evi bands are marked by arrows and unspecific bands by asterisks.
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 beta-catenin protein was used to assess MG13
* VAT and and shipping costs not included. Errors and price changes excepted
