Human Von Hippel Lindau / VHL ELISA Kit
- SKU:
- HUFI01668
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- P40337
- Sensitivity:
- 0.094ng/ml
- Range:
- 0.156-10ng/ml
- ELISA Type:
- Sandwich
- Synonyms:
- VHL, Von Hippel-Lindau disease tumor suppressor, Protein G7, pVHL, RCA1, VHL1, pVHL, HRCA1
- Reactivity:
- Human
- Research Area:
- Signal Transduction
Frequently bought together:
Description
Human Von Hippel Lindau / VHL ELISA
VHL (Von Hippel-Lindau Tumor Suppressor) is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign tumors. Mutations of VHL are associated with renal cell carcinoma, clear-cell sarcoma, and endolymphatic sac tumors. In normal cells, this protein product ubiquitinates hypoxia-inducible factor (HIF), targeting it for destruction via the proteasome under normoxic conditions. The VHL protein functions as a tumor suppressor by negatively regulating cellular responses to hypoxia.
| Product Name: | Human Von Hippel Lindau / VHL ELISA Kit |
| Product Code: | HUFI01668 |
| Size: | 96 Assays |
| Alias: | VHL, Von Hippel-Lindau disease tumor suppressor, Protein G7, pVHL, RCA1, VHL1, pVHL, HRCA1 |
| Detection method: | Sandwich ELISA, Double Antibody |
| Application: | This immunoassay kit allows for the in vitro quantitative determination of Human VHL concentrations in serum plasma and other biological fluids. |
| Sensitivity: | 0.094ng/ml |
| Range: | 0.156-10ng/ml |
| Storage: | 4°C for 6 months |
| Note: | For Research Use Only |
| Recovery: | Matrices listed below were spiked with certain level of Human VHL and the recovery rates were calculated by comparing the measured value to the expected amount of Human VHL in samples. | ||||||||||||||||
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| Linearity: | The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human VHL and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. | ||||||||||||||||
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| CV(%): | Intra-Assay: CV<8% Inter-Assay: CV<10% |
| Component | Quantity | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | 4°C for 6 months |
| Lyophilized Standard | 2 | 4°C/-20°C |
| Sample/Standard Dilution Buffer | 20ml | 4°C |
| Biotin-labeled Antibody(Concentrated) | 120ul | 4°C (Protect from light) |
| Antibody Dilution Buffer | 10ml | 4°C |
| HRP-Streptavidin Conjugate(SABC) | 120ul | 4°C (Protect from light) |
| SABC Dilution Buffer | 10ml | 4°C |
| TMB Substrate | 10ml | 4°C (Protect from light) |
| Stop Solution | 10ml | 4°C |
| Wash Buffer(25X) | 30ml | 4°C |
| Plate Sealer | 5 | - |
Other materials and equipment required:
- Microplate reader with 450 nm wavelength filter
- Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
- Incubator
- Deionized or distilled water
- Absorbent paper
- Buffer resevoir
| Uniprot | P40337 |
| UniProt Protein Function: | VHL: Involved in the ubiquitination and subsequent proteasomal degradation via the von Hippel-Lindau ubiquitination complex. Seems to act as target recruitment subunit in the E3 ubiquitin ligase complex and recruits hydroxylated hypoxia- inducible factor (HIF) under normoxic conditions. Involved in transcriptional repression through interaction with HIF1A, HIF1AN and histone deacetylases. Ubiquitinates, in an oxygen-responsive manner, ADRB2. Component of the VCB (VHL-Elongin BC-CUL2) complex; this complex acts as a ubiquitin-ligase E3 and directs proteasome- dependent degradation of targeted proteins. Interacts with CUL2; this interaction is dependent on the integrity of the trimeric VBC complex. Interacts (via the beta domain) with HIF1A (via the NTAD domain); this interaction mediates degradation of HIF1A in normoxia and, in hypoxia, prevents ubiqitination and degradation of HIF1A by mediating hypoxia-induced translocation to the nucleus, a process which requires a hypoxia-dependent regulatory signal. Interacts with ADRB2; the interaction, in normoxia, is dependent on hydroxylation of ADRB2 and the subsequent VCB- mediated ubiquitination and degradation of ADRB2. Under hypoxia, hydroxylation, interaction with VHL, ubiquitination and subsequent degradation of ADRB2 are dramatically decreased. Interacts with RNF139, USP33 and PHF17. Found in a complex composed of LIMD1, VHL, EGLN1/PHD2, TCEB2 AND CUL2. Isoform 1 and isoform 3 interact with LIMD1 (via LIM zinc-binding 2), AJUBA (via LIM domains) and WTIP (via LIM domains). Interacts with EPAS1. Expressed in the adult and fetal brain and kidney. 3 isoforms of the human protein are produced by alternative splicing. |
| UniProt Protein Details: | Protein type:Ubiquitin ligase; Tumor suppressor Chromosomal Location of Human Ortholog: 3p25.3 Cellular Component: nucleoplasm; intermediate filament cytoskeleton; mitochondrion; membrane; endoplasmic reticulum; cytosol; nucleus Molecular Function:protein binding; enzyme binding; ubiquitin-protein ligase activity; transcription factor binding Biological Process: negative regulation of cell proliferation; regulation of transcription, DNA-dependent; protein stabilization; positive regulation of transcription, DNA-dependent; cell morphogenesis; protein ubiquitination; negative regulation of transcription from RNA polymerase II promoter; proteolysis; positive regulation of cell differentiation; negative regulation of apoptosis Disease: Erythrocytosis, Familial, 2; Von Hippel-lindau Syndrome; Renal Cell Carcinoma, Nonpapillary; Pheochromocytoma |
| NCBI Summary: | Von Hippel-Lindau syndrome (VHL) is a dominantly inherited familial cancer syndrome predisposing to a variety of malignant and benign tumors. A germline mutation of this gene is the basis of familial inheritance of VHL syndrome. The protein encoded by this gene is a component of the protein complex that includes elongin B, elongin C, and cullin-2, and possesses ubiquitin ligase E3 activity. This protein is involved in the ubiquitination and degradation of hypoxia-inducible-factor (HIF), which is a transcription factor that plays a central role in the regulation of gene expression by oxygen. RNA polymerase II subunit POLR2G/RPB7 is also reported to be a target of this protein. Alternatively spliced transcript variants encoding distinct isoforms have been observed. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P40337 |
| NCBI GenInfo Identifier: | 4033778 |
| NCBI Gene ID: | 7428 |
| NCBI Accession: | P40337.2 |
| UniProt Secondary Accession: | P40337,Q13599, Q6PDA9, B2RE45, |
| UniProt Related Accession: | P40337 |
| Molecular Weight: | Calculated: 18kDa/19kDa/24kDaObserved: 24kDa |
| NCBI Full Name: | Von Hippel-Lindau disease tumor suppressor |
| NCBI Synonym Full Names: | von Hippel-Lindau tumor suppressor, E3 ubiquitin protein ligase |
| NCBI Official Symbol: | VHL |
| NCBI Official Synonym Symbols: | RCA1; VHL1; pVHL; HRCA1 |
| NCBI Protein Information: | von Hippel-Lindau disease tumor suppressor; protein G7; elongin binding protein |
| UniProt Protein Name: | Von Hippel-Lindau disease tumor suppressor |
| UniProt Synonym Protein Names: | Protein G7; pVHL |
| Protein Family: | Von Hippel-Lindau disease tumor suppressor |
| UniProt Gene Name: | VHL |
| UniProt Entry Name: | VHL_HUMAN |
*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.
| Step | Protocol |
| 1. | Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells! |
| 2. | Aliquot 0.1ml standard solutions into the standard wells. |
| 3. | Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well. |
| 4. | Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells. |
| 5. | Seal the plate with a cover and incubate at 37 °C for 90 min. |
| 6. | Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2. |
| 7. | Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall. |
| 8. | Seal the plate with a cover and incubate at 37°C for 60 min. |
| 9. | Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash. |
| 10. | Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min. |
| 11. | Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min. |
| 12. | Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color. |
| 13. | Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately. |
| 14. | Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution. |
When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.
| Sample Type | Protocol |
| Serum | If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. |
| Plasma | Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit. |
| Urine & Cerebrospinal Fluid | Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid. |
| Cell culture supernatant | Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately. |
| Cell lysates | Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Tissue homogenates | The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C. |
| Tissue lysates | Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C. |
| Breast Milk | Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles. |
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