Description
Human Nucleoside diphosphate kinase A (NME1) ELISA Kit
The Human Nucleoside Diphosphate Kinase A (NME1) ELISA Kit is a valuable tool for researchers looking to accurately measure NME1 levels in human samples such as serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, providing reliable and reproducible results for a variety of research applications.NME1, also known as nucleoside diphosphate kinase A, is an enzyme that plays a crucial role in regulating cell proliferation, migration, and differentiation.
Dysregulation of NME1 has been implicated in various diseases, including cancer, where it is often downregulated, highlighting its importance as a potential biomarker for disease diagnosis and prognosis.By using the Human Nucleoside Diphosphate Kinase A (NME1) ELISA Kit, researchers can gain valuable insights into the role of NME1 in disease pathology and progression, paving the way for the development of targeted therapies and personalized treatment options.
| Product Name: | Human Nucleoside diphosphate kinase A (NME1) ELISA Kit |
| SKU: | HUEB1518 |
| Size: | 96T |
| Target: | Human Nucleoside diphosphate kinase A (NME1) |
| Synonyms: | Granzyme A-activated DNase, Metastasis inhibition factor nm23, NM23-H1, Tumor metastatic process-associated protein, GAAD, NDK A, NDPKA, NM23 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.156-10ng/mL |
| Sensitivity: | 0.056ng/ml |
| Intra CV: | 3.8% | ||||||||||||||||||||
| Inter CV: | 7.9% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Major role in the synthesis of nucleoside triphosphates other than ATP. The ATP gamma phosphate is transferred to the NDP beta phosphate via a ping-pong mechanism, using a phosphorylated active-site intermediate. Possesses nucleoside-diphosphate kinase, serine/threonine-specific protein kinase, geranyl and farnesyl pyrophosphate kinase, histidine protein kinase and 3'-5' exonuclease activities. Involved in cell proliferation, differentiation and development, signal transduction, G protein-coupled receptor endocytosis, and gene expression. Required for neural development including neural patterning and cell fate determination. During GZMA-mediated cell death, works in concert with TREX1. NME1 nicks one strand of DNA and TREX1 removes bases from the free 3' end to enhance DNA damage and prevent DNA end reannealing and rapid repair. |
| Uniprot: | P15531 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Nucleoside diphosphate kinase A |
| Sub Unit: | Hexamer of two different chains: A and B (A6, A5B, A4B2, A3B3, A2B4, AB5, B6). Interacts with PRUNE. Component of the SET complex, composed of at least ANP32A, APEX1, HMGB2, NME1, SET and TREX1. Within this complex, interacts directly with SET. Also interacts with TREX1, but only following translocation to the nucleus. |
| Research Area: | Cancer |
| Subcellular Location: | Cytoplasm Nucleus Cell-cycle dependent nuclear localization which can be induced by interaction with Epstein-barr viral proteins or by degradation of the SET complex by GzmA. |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | NME1: an enzyme with multiple activities including histidine protein kinase, nucleoside-diphosphate kinase, serine/threonine-specific protein kinase, geranyl and farnesyl pyrophosphate kinase and 3'-5' exonuclease activities. Plays a major role in the synthesis of nucleoside triphosphates other than ATP. The ATP gamma phosphate is transferred to the NDP beta phosphate via a ping-pong mechanism, using a phosphorylated active-site intermediate. Involved in cell proliferation, differentiation and development, G protein-coupled receptor endocytosis, cytotoxic T cell and NK cell-mediated cell death, and gene expression. Required for neural development including neural patterning and cell fate determination. During granzyme A (GZMA)-mediated cell death, works in concert with TREX1. NME1 nicks one strand of DNA and TREX1 removes bases from the free 3' end to enhance DNA damage and prevent DNA end reannealing and rapid repair. Three alternatively spliced human proteins have been reported. |
| UniProt Protein Details: | Protein type:Kinase, other; EC 2.7.4.6; Tumor suppressor; Nucleotide Metabolism - pyrimidine; Kinase, nucleoside diphosphate; Protein kinase, histidine; Nucleotide Metabolism - purine; Other group; NDK family Chromosomal Location of Human Ortholog: 17q21.3 Cellular Component: cytoplasm; cytosol; membrane; nucleus Molecular Function:ATP binding; deoxyribonuclease activity; GTP binding; identical protein binding; magnesium ion binding; nucleoside diphosphate kinase activity; protein binding; ribosomal small subunit binding Biological Process: negative regulation of cell proliferation; nucleobase, nucleoside and nucleotide interconversion; nucleoside triphosphate biosynthetic process; positive regulation of DNA binding; positive regulation of epithelial cell proliferation; purine nucleotide metabolic process; pyrimidine nucleotide metabolic process; regulation of apoptosis Disease: Neuroblastoma, Susceptibility To |
| NCBI Summary: | This gene (NME1) was identified because of its reduced mRNA transcript levels in highly metastatic cells. Nucleoside diphosphate kinase (NDK) exists as a hexamer composed of 'A' (encoded by this gene) and 'B' (encoded by NME2) isoforms. Mutations in this gene have been identified in aggressive neuroblastomas. Two transcript variants encoding different isoforms have been found for this gene. Co-transcription of this gene and the neighboring downstream gene (NME2) generates naturally-occurring transcripts (NME1-NME2), which encodes a fusion protein comprised of sequence sharing identity with each individual gene product. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P15531 |
| NCBI GenInfo Identifier: | 127981 |
| NCBI Gene ID: | 4830 |
| NCBI Accession: | P15531.1 |
| UniProt Secondary Accession: | P15531,Q6FGK3, Q86XQ2, Q9UDJ6, |
| UniProt Related Accession: | P15531 |
| Molecular Weight: | 30,137 Da |
| NCBI Full Name: | Nucleoside diphosphate kinase A |
| NCBI Synonym Full Names: | NME/NM23 nucleoside diphosphate kinase 1 |
| NCBI Official Symbol: | NME1 |
| NCBI Official Synonym Symbols: | NB; AWD; NBS; GAAD; NDKA; NM23; NDPKA; NDPK-A; NM23-H1 |
| NCBI Protein Information: | nucleoside diphosphate kinase A |
| UniProt Protein Name: | Nucleoside diphosphate kinase A |
| UniProt Synonym Protein Names: | Granzyme A-activated DNase; GAAD; Metastasis inhibition factor nm23; NM23-H1; Tumor metastatic process-associated protein |
| Protein Family: | Nucleoside diphosphate kinase |
| UniProt Gene Name: | NME1 |
| UniProt Entry Name: | NDKA_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. |
