Description
Human Ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) ELISA Kit
The Human Ectonucleoside Triphosphate Diphosphohydrolase 5 (ENTPD5) ELISA Kit is a powerful tool for accurately measuring ENTPD5 levels in human biological samples such as serum, plasma, and cell culture supernatants. This kit is highly sensitive and specific, ensuring precise and reliable results for a variety of research purposes.ENTPD5 is a key enzyme involved in nucleotide metabolism and has been implicated in various diseases such as cancer, inflammatory disorders, and autoimmune diseases.
By measuring ENTPD5 levels, researchers can gain valuable insights into the role of this enzyme in disease progression and potentially identify new therapeutic targets.Overall, the ENTPD5 ELISA Kit from Assay Genie is an essential tool for researchers studying nucleotide metabolism and its impact on human health, providing accurate and reliable data for a wide range of research applications.
| Product Name: | Human Ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) ELISA Kit |
| SKU: | HUEB2076 |
| Size: | 96T |
| Target: | Human Ectonucleoside triphosphate diphosphohydrolase 5 (ENTPD5) |
| Synonyms: | CD39 antigen-like 4, ER-UDPase, Guanosine-diphosphatase ENTPD5, Nucleoside diphosphatase, Uridine-diphosphatase ENTPD5, GDPase ENTPD5, UDPase ENTPD5, NTPDase 5, CD39L4, PCPH |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.78-50ng/mL |
| Sensitivity: | 0.404ng/mL |
| Intra CV: | Provided with the Kit |
| Inter CV: | Provided with the Kit |
| Linearity: | Provided with the Kit |
| Recovery: | Provided with the Kit |
| Function: | Uridine diphosphatase (UDPase) that promotes protein N-glycosylation and ATP level regulation. UDP hydrolysis promotes protein N-glycosylation and folding in the endoplasmic reticulum, as well as elevated ATP consumption in the cytosol via an ATP hydrolysis cycle. Together with CMPK1 and AK1, constitutes an ATP hydrolysis cycle that converts ATP to AMP and results in a compensatory increase in aerobic glycolysis. The nucleotide hydrolyzing preference is GDP > IDP > UDP, but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. Plays a key role in the AKT1-PTEN signaling pathway by promoting glycolysis in proliferating cells in response to phosphoinositide 3-kinase (PI3K) signaling. |
| Uniprot: | O75356 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Ectonucleoside triphosphate diphosphohydrolase 5 |
| Sub Unit: | Exists both as a monomer and a disulfide-linked homodimer, the dimers are enzymatically inactive. |
| Research Area: | Cancer |
| Subcellular Location: | Endoplasmic reticulum Secreted |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | ENTPD5: Uridine diphosphatase (UDPase) that promotes protein N- glycosylation and ATP level regulation. UDP hydrolysis promotes protein N-glycosylation and folding in the endoplasmic reticulum, as well as elevated ATP consumption in the cytosol via an ATP hydrolysis cycle. Together with CMPK1 and AK1, constitutes an ATP hydrolysis cycle that converts ATP to AMP and results in a compensatory increase in aerobic glycolysis. Also hydrolyzes GDP and IDP but not any other nucleoside di-, mono- or triphosphates, nor thiamine pyrophosphate. Plays a key role in the AKT1-PTEN signaling pathway by promoting glycolysis in proliferating cells in response to phosphoinositide 3-kinase (PI3K) signaling. Belongs to the GDA1/CD39 NTPase family. |
| UniProt Protein Details: | Protein type:Hydrolase; Nucleotide Metabolism - pyrimidine; Oncoprotein; EC 3.6.1.6; EC 3.6.1.42; Nucleotide Metabolism - purine Chromosomal Location of Human Ortholog: 14q24 Cellular Component: endoplasmic reticulum Molecular Function:guanosine-diphosphatase activity; protein binding; uridine-diphosphatase activity Biological Process: 'de novo' posttranslational protein folding; ATP metabolic process; cell growth; cell proliferation; positive regulation of glycolysis; protein amino acid N-linked glycosylation; regulation of phosphoinositide 3-kinase cascade |
| NCBI Summary: | The protein encoded by this gene is similar to E-type nucleotidases (NTPases)/ecto-ATPase/apyrases. NTPases, such as CD39, mediate catabolism of extracellular nucleotides. ENTPD5 contains 4 apyrase-conserved regions which is characteristic of NTPases. [provided by RefSeq, Jan 2009] |
| UniProt Code: | O75356 |
| NCBI GenInfo Identifier: | 18202142 |
| NCBI Gene ID: | 957 |
| NCBI Accession: | O75356.1 |
| UniProt Secondary Accession: | O75356,Q96RX0, A1L4C5, |
| UniProt Related Accession: | O75356 |
| Molecular Weight: | 47,517 Da |
| NCBI Full Name: | Ectonucleoside triphosphate diphosphohydrolase 5 |
| NCBI Synonym Full Names: | ectonucleoside triphosphate diphosphohydrolase 5 |
| NCBI Official Symbol: | ENTPD5 |
| NCBI Official Synonym Symbols: | PCPH; CD39L4; NTPDase-5 |
| NCBI Protein Information: | ectonucleoside triphosphate diphosphohydrolase 5 |
| UniProt Protein Name: | Ectonucleoside triphosphate diphosphohydrolase 5 |
| UniProt Synonym Protein Names: | CD39 antigen-like 4; ER-UDPase; Guanosine-diphosphatase ENTPD5 (EC:3.6.1.42); GDPase ENTPD5; Nucleoside diphosphatase; Uridine-diphosphatase ENTPD5; UDPase ENTPD5 |
| Protein Family: | Ectonucleoside triphosphate diphosphohydrolase |
| UniProt Gene Name: | ENTPD5 |
| UniProt Entry Name: | ENTP5_HUMAN |
| Component | Quantity (96 Assays) | Storage |
| ELISA Microplate (Dismountable) | 8×12 strips | -20°C |
| Lyophilized Standard | 2 | -20°C |
| Sample Diluent | 20ml | -20°C |
| Assay Diluent A | 10mL | -20°C |
| Assay Diluent B | 10mL | -20°C |
| Detection Reagent A | 120µL | -20°C |
| Detection Reagent B | 120µL | -20°C |
| Wash Buffer | 30mL | 4°C |
| Substrate | 10mL | 4°C |
| Stop Solution | 10mL | 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
*Note: The below protocol is a sample protocol. Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.
Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.
| Step | |
| 1. | Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C. |
| 2. | Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform. |
| 3. | Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper. |
| 4. | Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C. |
| 5. | Repeat the wash process for five times as conducted in step 3. |
| 6. | Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction. |
| 7. | Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing. |
| 8. | Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters. |
| 9. | After experiment, store all reagents according to the specified storage temperature respectively until their expiry. |
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. |
