Mouse V5-TAG Monoclonal Antibody | anti-V5-TAG antibody

MOUSE ANTI V5-TAG

Cat. Num. AAA212109

• Applications: Immunohistochemistry, ELISA, Flow Cytometry, Functional Assay, Immunofluorescence, Immunoprecipitation, Western Blot, immunoassay, Radioimmunoassay
• Synonyms: V5-TAG; Monoclonal Antibody; MOUSE ANTI V5-TAG; anti-V5-TAG antibody

• Host: Mouse
• Clonality: Monoclonal
• Isotype: IgG2a
• Clone Number: SV5-Pk1
• Form/Format: Purified; Purified IgG - liquid
• Concentration: IgG concentration 1 mg/ml (varies by lot)
• Sequence Length: 222

• Applicable Applications for anti-V5-TAG antibody: Immunohistology Frozen, ELISA (EIA), Flow cytometry (FC/FACS), Immunofluorescence (IF), Immunoprecipitation (IP), P, Western Blot (WB), Radioimmunoassays (RIA)
• Application Notes: ELISA: Minimum Dilution: 1/1000; Maximum Dilution: 1/5000;; Western Blotting: Minimum Dilution: 1/1000; Maximum Dilution: 1/5000
• Perservative Stabilisers: 0.09% Sodium Azide (NaN3) ; Preparation
• Buffer Solution: Phosphate buffered saline
• Target Species: Viral
• Preparation and Storage: Store at 4 degree C or at -20 degree C if preferred. This product should be stored undiluted. Storage in frost-free freezers is not recommended. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use.; Shelf Life: 18 months from date of despatch.

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged REDD1 protein by western blotting.Image caption:REDD1 is not degraded by Cul4a or phosphorylated by GSK3beta at Thr 23 and Thr25. (A) HEK293 cells were transfected with empty vector or tetracycline inducible dnCul4a-V5 pcDNA4/TO (1 ug) and Cul4A-V5 pcDNA3 (0.03 ug) followed by tetracycline (1 ug/ml) induction for 24 hours and cell lysis. (B) HEK293 cells were transfected with 0.5 ug Cul4a-V5 pcDNA3 for 15 hours followed by transfection of 20 nM control or Cul4a siRNAs to determine siRNAs efficiency. (C) HEK293 cells were transfected with 20 nM control or Cul4a siRNAs for 3 days followed by cell lysis. (D) REDD1-V5 pcDNA3 wild type, T23A T25A or T23D T25D plasmids (0.4 ug) were transfected in HEK293 cells for 3 days and treated with 20 uM MG-132 for 6 hours followed by cell lysis. (E) HEK293 cells were treated with 30 mM LiCl or GSK3 inhibitor IX (5 uM or 10 uM) for 20 hours followed by cell lysis. (F) HEK293 cells were co-transfected with 0.2 ug REDD1-V5 pcDNA3 and 0.3 ug GSK3beta pcDNA3 or empty pcDNA3 for 3 days followed by MG-132 (20 uM) treatment for 6 hours followed by cell lysis. (G) HEK293 cells were transfected with 3 ug DYKDDDDK-REDD1 or DYKDDDDK-FRAT1 for 3 days followed by cell lysis and DYKDDDDK immunoprecipitation. In vitro phosphorylation of REDD1 and FRAT1 was carried out as described in Materials and Methods..From: Tan CY, Hagen T (2013) mTORC1 Dependent Regulation of REDD1 Protein Stability. PLoS ONE 8(5): e63970.)

Testing Data

(Published customer image: Mouse anti V5 Tag antibody, clone SV5-Pk1used for immunoprecipitation of V5 tagged RNF11 proteinImage caption:Myristoylation mutant of RNF11 is unable to associate with Itch. SH-SY5Y shRNA-RNF11 cells were transfected with shRNA-resistant RNF11 constructs or vector. Coimmunoprecipitation experiments using V5 antibody were performed 24 hours after transfection. Immunoprecipitates and lysates were resolved by SDS-PAGE and immunoblotted with anti-A20, Itch, RNF11 or actin. Blots are representative of three independent experiments. From: Pranski EL, Dalal NV, Herskowitz JH, Orr AL, Roesch LA, Fritz JJ, Heilman C, Lah JJ, Levey AI, Betarbet RS. Neuronal RING finger protein 11 (RNF11) regulates canonical NF-?B signaling. J Neuroinflammation. 2012 Apr 16;9:67. doi: 10.1186/1742-2094-9-67.)

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for the detection of V5 tagged HIVA peptide by western blottingImage caption:Construction of the BCG.HIVACAT vaccine strain. (A) A synthetic GC-rich HIVA gene was fused to the region encoding the 19-kDa lipoprotein signal sequence and inserted into the episomal pJH222 E Coli-mycobacterium shuttle plasmid. This plasmid contains kanamycin resistance (aph) and complementing lysA genes and an E Coli origin of replication (oriE). In addition, pJH222 contained the mycobacterial origin of replication (oriM). The BALB/c mouse T-cell and MAb Pk epitopes used in this work are depicted. P a-Ag, M. tuberculosis a-antigen promoter; PHSP60, heat shock protein 60 gene promoter. The aph gene was removed by SpeI digestion and the lacO sequence was inserted and transformed into E Coli DH1lacdapD strain. (B) Immunodot of BCG.HIVACAT lysates. Dot 1: BCG wild type (negative control); Dot 2, 3, 4 and 5: clone 3, clone 7, clone 9 and clone 10 of BCG.HIVACAT; Dot 6: BCG.HIVA222 (positive control). HIVA peptide was detected using the anti-Pk MAb followed by horseradish peroxidase-Goat-anti-Mouse and enhanced chemiluminescence (ECL) detection. (C) In vivo plasmid stability of BCG.HIVACAT harboring pJH222.HIVACAT. Mice were injected s.c. with 105 cfu of BCG.HIVACAT and boosted i.m. with 106 pfu of MVA.HIVA, spleens were homogenized 20 weeks after BCG inoculation and the recovered rBCG colonies were tested for the presence of the HIVA DNA coding sequence by PCR. Lanes 1 to 6: Six rBCG colonies were recovered in the non-lysine supplemented plate; lane 7: Molecular weight marker; lane 8: Plasmid DNA positive control; lane 9: Distilled water (negative control).From: Saubi N, Mbewe-Mvula A, Gea-Mallorqui E, Rosario M, Gatell JM, et al. (2012) Pre-Clinical Development of BCG.HIVACAT, an Antibiotic-Free Selection Strain, for HIV-TB Pediatric Vaccine Vectored by Lysine Auxotroph of BCG. PLoS ONE 7(8): e42559.)

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged REDD1 protein by western blotting.Image caption:REDD1 is not regulated by Cullin E3 Ubiquitin ligases. (A,B) Untransfected (A) or REDD1-V5 pcDNA3 (0.3 ug) transfected (B) HEK293 cells stably expressing tetracycline inducible dnUbc12-HA were induced with 1 ug/ml tetracycline for 24 hours (A,B) or treated with 3 uM MLN4924 (A) or 20 uM MG-132 (A,B) for 8 hours followed by cell lysis. (C,D) Untransfected HEK293 (C) or HEK293 transfected with REDD1-V5 pcDNA3 (0.3 ug) (D) were pre-treated with 3 uM MLN4924 followed by cycloheximide (40 uM) treatment and cell lysis at the indicated time points. (E) HEK293 cells stably expressing tetracycline inducible dnUbc12-HA were induced with 1 ug/ml tetracycline for 24 hours followed by cycloheximide (40 uM) treatment and cell lysis at the indicated time points.From: Tan CY, Hagen T (2013) mTORC1 Dependent Regulation of REDD1 Protein Stability. PLoS ONE 8(5): e63970.)

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged podocin protein by western blottingImage caption:The podocin short isoform interacts with known podocyte proteins. CD2AP, TRPC6, neprin and NEPH1 co-precipitate with both podocin isoforms (FL, full length (canoncical isoform); SI, short isoform). DYKDDDDK- and V5-tagged proteins were expressed in HEK293T cells and precipitated with anti-DYKDDDDK antibody as indicated. Western blot analysis was performed with a V5 specific antibody. Expression levels of DYKDDDDK.podocin constructs in the lysates are shown below.From: V¶lker LA, Schurek EM, Rinschen MM, Tax J, Schutte BA, Lamkemeyer T, Ungrue D, Schermer B, Benzing T, H¶hne M. Characterization of a short isoform of the kidney protein podocin in human kidney. BMC Nephrol. 2013 May 6;14:102.)

Testing Data

(V5 tagged protein detected with Mouse anti V5-Tag)

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for detection of V5 tagged podocin protein by western blottingImage caption:The podocin short isoform is N-glycosylated. A) PNGase-F treatment removes the double band from the short isoform in DRM fraction 7. Lysates from Figure 3 were subjected to treatment with PNGase-F and immunoblotted and detected with anti-V5 antibody. B) N to S mutation of the N-glycosylation consensus motif completely abrogates the formation of a double band. The asparagine at position 287 corresponds to amino acid 355 in the full-length protein. HEK293T cells were transfected with V5-tagged Podocin (short isoform or short isoform N287S, respectively) and lysates were immunoblotted and detected with anti-V5 antibody.From: V¶lker LA, Schurek EM, Rinschen MM, Tax J, Schutte BA, Lamkemeyer T, Ungrue D, Schermer B, Benzing T, H¶hne M. Characterization of a short isoform of the kidney protein podocin in human kidney. BMC Nephrol. 2013 May 6;14:102.)

Testing Data

(Published customer image: Mouse anti V5 Tag antibody, clone SV5-Pk1used for immunoprecipitation of V5 tagged RNF11 proteinImage caption:Dynamic associations of RNF11 with both A20 and Itch in primary neurons. N2A cells transduced with V5-RNF11 lentivirus (N2A V5-RNF11) and transfected with DYKDDDDK-A20 were harvested for immunoprecipitation (IP) with V5 antibody (A) or harvested for IP with DYKDDDDK antibody (B). Proteins were resolved by SDS-PAGE and immunoblotted with anti-A20 and RNF11. In parallel, pull-down assays with V5 antibody (C) or with Itch antibody (D) from N2A V5-RNF11 cell lysates were resolved by SDS-PAGE. Immunoprecipitates and lysates were immunoblotted with anti-Itch and RNF11. (E) and (H) Murine primary cortical neurons were stimulated with 10?ng/ml TNF-a for 0 or 30 minutes and harvested for IP with RNF11 antibody. Control IP experiments were performed with antibody omitted. Proteins were resolved by SDS-PAGE and immunoblotted with anti-A20, Itch, RNF11 and actin. (F), (G), (I) and (J) ImageJ software was used to quantify the densitometry of the immunoprecipitated bands relative to the 0-minutes time point. Each input sample's immunoreactivity was used as a loading control. All IPs are representative of at least three independent experiments. *P?)

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for the detection of V5 tagged Rho GTPase-activating protein 28 protein by western blotting and immunofluorescenceImage caption:Arhgap28-V5 inhibits RhoA activation and stress fiber formation in SaOS-2 cells. SaOS-2 cells were transfected with empty vector or Arhgap28-V5. A. The expression of Arhgap28-V5 was confirmed by western blotting using an antibody to V5. B. Effect of Arhgap28-V5 expression on the basal activity of RhoA (n = 5), Rac1 (n = 3) and Cdc42 (n = 3). Bars show SEM. * indicates significant difference found, p)

Testing Data

(Published customer image: Mouse anti V5 tag antibody, clone SV5-Pk1 used for the detection of V5 tagged WEEV_nsP3 protein by western blotting and immunofluorescenceImage caption: WEEV nsP3 interaction with host IKKbeta. A) U87MGs were transfected in a 6-well plate with 5 ug of pUC19 and WEEV_nsP3_HA for 24 hours. Cell lysates were resolved using SDS-PAGE and subsequently immunoblotted with V5 antibody and beta-actin served as a loading control. B) U87MGs were transfected with WEEV_nsP3_V5; cells were fixed after 24 hours and stained with antibodies against the endogenous IKKbeta and the V5 tag. Cells were incubated with appropriate secondary Alexa Fluor antibodies and the nuclei stained with DAPI. Co-localization of IKKbeta with WEEV_nsP3_V5 (yellow) was observed as shown by the arrows. B) Panels E -H serve as an example of transfected cells in a given field of view that show co-localization of IKKbeta and WEEV_nsP3_V5 24 hours post transfection. Panels I-L represent magnified images of other cells showing co-localization of IKKbeta and WEEV_nsP3_V5. Panel M is a magnified image of panel L. The co-localization was confirmed by Z-stack analysis. Co-localization was calculated to be approximately in 61% of cells (163 cells were counted of which 44% demonstrated expression of nsP3. Of those cells that expressed nsP3, 61% showed co-localization of both proteins). Images were taken using Nikon Eclipse TE2000-U at 60x magnification and are representative of 2 independent experiments.From: Amaya M, Voss K, Sampey G, Senina S, de la Fuente C, et al. (2014) The Role of IKKbeta in Venezuelan Equine Encephalitis Virus Infection. PLoS ONE 9(2): e86745.)

NCBI and Uniprot Product Information

• NCBI GI #: 55775699
• NCBI GeneID: 3160800
• NCBI Accession #: YP_138513.1
• NCBI GenBank Nucleotide #: NC_006430.1
• NCBI Official Full Name: V protein
• NCBI Official Symbol: V/P
• NCBI Protein Information: phosphoprotein; V protein