Western blot analysis of VHL expression and RNAi validation of anti-VHL antibody specificity - Panel 1. Lysate prepared from HeLa S3 cells (Panel 1a) was blotted using Purified Mouse Anti-VHL antibody (Cat. No. 564183) at concentrations of 1 (lane 1), 0.5 (lane 2), or 0.25 (lane 3) μg/ml. VHL was identified as a band of ~24 kDa (Panel 1a). Lysates from untreated, VHL RNAi- or ERK2 RNAi-treated HeLa S3 cells (Panel 1b) were blotted with Purified Mouse Anti-VHL or Anti-ERK-2 (Cat. No. 554095) antibody. The expressed level of VHL protein blotted with Anti-VHL antibody (2 μg/ml) is downregulated in lysate prepared from HeLa S3 cells treated with VHL RNAi but not ERK2 RNAi. Similarly, ERK2 protein expression was reduced in lysate from HeLa S3 cells treated with ERK2 RNAi but not VHL RNAi when blotted with Anti-ERK2 antibody (0.5 μg/ml).
Immunohistochemical staining of VHL expressed in tissue from a human renal tumor - Panel 2. Following antigen retrieval with BD Retrievagen A buffer (Cat. no. 550524), the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b, κ Isotype Control (Cat. No. 557351; Top Tissue) or Purified Mouse Anti-VHL antibody (Bottom Tissue) using the BD Biosciences Protocol for Immunohistochemical Staining. A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin (Cat. No. 550337), Streptavidin-Horseradish Peroxidase (HRP) (Cat. No.550946), and the DAB Substrate Kit (Cat. No. 550880) was used to develop the primary staining reagents. The tissue was counterstained with Hematoxylin. Original magnification: 20X.
Flow cytometric analysis of VHL expression in VHL-transfected 293F cells - Panel 3. Untransfected (dashed line histogram) and human VHL-transfected (solid line histogram) 293F cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then washed and stained with Purified Mouse Anti-Human VHL antibody followed by APC Goat Anti-Mouse Ig (Cat. No. 550826) using BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD FACSCanto™ II Flow Cytometer System.
Western blot analysis of VHL expression and RNAi validation of anti-VHL antibody specificity - Panel 1. Lysate prepared from HeLa S3 cells (Panel 1a) was blotted using Purified Mouse Anti-VHL antibody (Cat. No. 564183) at concentrations of 1 (lane 1), 0.5 (lane 2), or 0.25 (lane 3) μg/ml. VHL was identified as a band of ~24 kDa (Panel 1a). Lysates from untreated, VHL RNAi- or ERK2 RNAi-treated HeLa S3 cells (Panel 1b) were blotted with Purified Mouse Anti-VHL or Anti-ERK-2 (Cat. No. 554095) antibody. The expressed level of VHL protein blotted with Anti-VHL antibody (2 μg/ml) is downregulated in lysate prepared from HeLa S3 cells treated with VHL RNAi but not ERK2 RNAi. Similarly, ERK2 protein expression was reduced in lysate from HeLa S3 cells treated with ERK2 RNAi but not VHL RNAi when blotted with Anti-ERK2 antibody (0.5 μg/ml).
Immunohistochemical staining of VHL expressed in tissue from a human renal tumor - Panel 2. Following antigen retrieval with BD Retrievagen A buffer (Cat. no. 550524), the formalin-fixed paraffin-embedded sections were stained with either Purified Mouse IgG2b, κ Isotype Control (Cat. No. 557351; Top Tissue) or Purified Mouse Anti-VHL antibody (Bottom Tissue) using the BD Biosciences Protocol for Immunohistochemical Staining. A three-step staining procedure that employs a Biotin Goat Anti-Mouse Immunoglobulin (Cat. No. 550337), Streptavidin-Horseradish Peroxidase (HRP) (Cat. No.550946), and the DAB Substrate Kit (Cat. No. 550880) was used to develop the primary staining reagents. The tissue was counterstained with Hematoxylin. Original magnification: 20X.
Flow cytometric analysis of VHL expression in VHL-transfected 293F cells - Panel 3. Untransfected (dashed line histogram) and human VHL-transfected (solid line histogram) 293F cells were fixed with BD Cytofix™ Fixation Buffer (Cat. No. 554655) and permeabilized with BD Phosflow™ Perm Buffer III (Cat. No. 558050). The cells were then washed and stained with Purified Mouse Anti-Human VHL antibody followed by APC Goat Anti-Mouse Ig (Cat. No. 550826) using BD Biosciences Protocol for Intracellular Staining. The fluorescence histograms were derived from gated events with the forward and side light-scatter characteristics of intact cells. Flow cytometric analysis was performed using a BD FACSCanto™ II Flow Cytometer System.
Product Details
BD Pharmingen™
VHL1; pVHL; Elongin binding protein; Protein G7; HRCA1; RCA1
Human (QC Testing)
Mouse IgG2b, κ
Human VHL Recombinant Protein
Intracellular staining (flow cytometry) (Routinely Tested), Immunohistochemistry-formalin (antigen retrieval required), Western blot (Tested During Development)
23-25 kDa
0.5 mg/ml
AB_2738651
Aqueous buffered solution containing ≤0.09% sodium azide.
RUO
Preparation And Storage
The monoclonal antibody was purified from tissue culture supernatant or ascites by affinity chromatography. Store undiluted at 4°C.
Product Notices
- Please refer to www.bdbiosciences.com/us/s/resources for technical protocols.
- Since applications vary, each investigator should titrate the reagent to obtain optimal results.
- Sodium azide is a reversible inhibitor of oxidative metabolism; therefore, antibody preparations containing this preservative agent must not be used in cell cultures nor injected into animals. Sodium azide may be removed by washing stained cells or plate-bound antibody or dialyzing soluble antibody in sodium azide-free buffer. Since endotoxin may also affect the results of functional studies, we recommend the NA/LE (No Azide/Low Endotoxin) antibody format, if available, for in vitro and in vivo use.
- Caution: Sodium azide yields highly toxic hydrazoic acid under acidic conditions. Dilute azide compounds in running water before discarding to avoid accumulation of potentially explosive deposits in plumbing.
- For fluorochrome spectra and suitable instrument settings, please refer to our Multicolor Flow Cytometry web page at www.bdbiosciences.com/colors.
Companion Products
Stain Buffer (FBS) RUO
Size
500 mL
Cat No.
554656
Stain Buffer (BSA) RUO
Size
500 mL
Cat No.
554657
Fixation Buffer RUO
Size
100 mL
Cat No.
554655
Perm Buffer III RUO
Size
125 mL
Cat No.
558050
Retrievagen A (pH 6.0) RUO
Cat No.
550524
Purified Mouse IgG2b, κ Isotype Control RUO
Size
0.5 mg
Cat No.
557351
564183 Rev. 1
Antibody Details
S2-647
The S2-647 monoclonal antibody recognizes the Von Hippel-Lindau (VHL) protein. The VHL protein is expressed within the cytosol and nuclei of cells from a variety of tissues. It serves as a tumor suppressor and transcriptional repressor that is highly conserved from Drosophila to mammalian species. VHL binds to Elongin B and C subunits and other proteins as part of a E3 ubiquitin ligase complex that targets a variety of proteins, including HIF-1 alpha and Beta-2 adrenergic receptors, for proteasomal degradation. Aberrant VHL expression is associated with a number of diseases including Von Hippel-Lindau disease, a familial cancer syndrome, that predisposes individuals to hemangioblastomas of the central nervous system and retina, pheochromocytoma and renal cell carcinoma. Three distinct VHL isoforms ranging in molecular weight from approximately 19-30 kDa have been described.
564183 Rev. 1
Format Details
Purified
Tissue culture supernatant is purified by either protein A/G or affinity purification methods. Both methods yield antibody in solution that is free of most other soluble proteins, lipids, etc. This format provides pure antibody that is suitable for a number of downstream applications including: secondary labeling for flow cytometry or microscopy, ELISA, Western blot, etc.
Purified
564183 Rev.1
Citations & References
Development References (8)
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Baba M, Hirai S, Kawakam S, et al. Tumor suppressor protein VHL is induced at high cell density and mediates contact inhibition of cell growth. Oncogene. 2001; 20(22):2727-2736. (Biology). View Reference
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Chen F, Kishida T, Duh FM, et al. Suppression of growth of renal carcinoma cells by the von Hippel-Lindau tumor suppressor gene. Cancer Res. 1995; 55(21):4804-4807. (Biology). View Reference
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Hsu T, Adereth Y, Kose N, Dammai V. Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility. J Biol Chem. 2006; 28(281):12069-12080. (Biology). View Reference
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Iliopoulos O, Kibel A, Gray S, Kaelin WG Jr. Tumour suppression by the human von Hippel-Lindau gene product.. Nat Med. 1995; 1(8):822-826. (Biology). View Reference
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Iliopoulos O, Ohh M, Kaelin WG Jr.. pVHL19 is a biologically active product of the von Hippel-Lindau gene arising from internal translation initiation. Proc Natl Acad Sci U S A. 1998; 95(20):11661-11666. (Biology). View Reference
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In: Hesketh R. The Oncogene Handbook. New York: Academic Press; 1994:559.
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Kibel A, Iliopoulos O, DeCaprio JA, Kaelin WG Jr. Binding of the von Hippel-Lindau tumor suppressor protein to Elongin B and C. Science. 1995; 269(5229):1444-1446. (Biology). View Reference
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Qi H, Gervais ML, Li W, DeCaprio JA, Challis JR, Ohh M. Molecular cloning and characterization of the von Hippel-Lindau-like protein. Mol Cancer Res. 2004; 2(1):43-52. (Biology). View Reference
564183 Rev. 1
Please refer to Support Documents for Quality Certificates
Global - Refer to manufacturer's instructions for use and related User Manuals and Technical data sheets before using this products as described
Comparisons, where applicable, are made against older BD Technology, manual methods or are general performance claims. Comparisons are not made against non-BD technologies, unless otherwise noted.
For Research Use Only. Not for use in diagnostic or therapeutic procedures.
