![Human Isocitrate dehydrogenase [NADP], mitochondrial (IDH2) ELISA Kit (HUEB1204)](https://cdn11.bigcommerce.com/s-h68l9z2lnx/images/stencil/1280x1280/products/35009/663704/human-isocitrate-dehydrogenase-nadp-mitochondrial-idh2-elisa-kit-hueb1204__75767.1745716240.jpg?c=2?imbypass=on "Human Isocitrate dehydrogenase [NADP], mitochondrial (IDH2) ELISA Kit (HUEB1204)")
Description
Human Isocitrate dehydrogenase [NADP], mitochondrial (IDH2) ELISA Kit
The Human Isocitrate Dehydrogenase NADP+ Mitochondrial (IDH2) ELISA Kit from Assay Genie is a powerful tool for detecting and quantifying levels of IDH2 in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit provides accurate and reliable results for researchers working in a variety of fields.IDH2 is a key enzyme involved in cellular metabolism, particularly in the citric acid cycle. Mutations in the IDH2 gene have been linked to various diseases, including cancer and metabolic disorders, making it a valuable biomarker for studying these conditions and potentially developing targeted therapies.
By using the Human Isocitrate Dehydrogenase NADP+ Mitochondrial (IDH2) ELISA Kit, researchers can gain valuable insights into the role of IDH2 in health and disease, paving the way for new discoveries and treatment options. Upgrade your research capabilities with this essential kit from Assay Genie.
| Product Name: | Human Isocitrate dehydrogenase [NADP], mitochondrial (IDH2) ELISA Kit |
| SKU: | HUEB1204 |
| Size: | 96T |
| Target: | Human Isocitrate dehydrogenase [NADP], mitochondrial (IDH2) |
| Synonyms: | ICD-M, IDP, NADP(+)-specific ICDH, Oxalosuccinate decarboxylase, IDH |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.312-20ng/mL |
| Sensitivity: | 0.098ng/mL |
| Intra CV: | 4.4% | ||||||||||||||||||||
| Inter CV: | 7.1% | ||||||||||||||||||||
| Linearity: |
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| Recovery: |
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| Function: | Plays a role in intermediary metabolism and energy production. It may tightly associate or interact with the pyruvate dehydrogenase complex. |
| Uniprot: | P48735 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Isocitrate dehydrogenase [NADP], mitochondrial |
| Sub Unit: | Homodimer. |
| Subcellular Location: | Mitochondrion |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | IDH2: a mitochondrial oxidoreductase that catalyzes the third step of the citric acid cycle: the oxidative decarboxylation of isocitrate, consuming NADP(+), and producing alpha-ketoglutarate (alpha-KG) and CO2. Can also use oxalosuccinate as a substrate. Alpha-KG is an activator the dioxygenases that hydroxylate the transcription factor HIF and lead to its degradation by VHL. Since HIF turns on oncogenic pathways, IDH2 has apparent tumor suppressor activity. Homo-dimerization is required for activity. Each subunit binds 1 magnesium or manganese ion. May tightly associate or interact with the pyruvate dehydrogenase complex. A somatic mutation resulting in the R172K conversion results in a neomorphic enzyme activity that converts isocitrate to R(-)-2-hydroxyglutarate (2HG) occurs in a significant percentage of patients with cytogenetically normal acute myeloid leukemia (AML). Elevated levels of 2HG are also correlated with an elevated risk of malignant brain tumors. |
| UniProt Protein Details: | Protein type:Mitochondrial; Carbohydrate Metabolism - citrate (TCA) cycle; EC 1.1.1.42; Other Amino Acids Metabolism - glutathione; Oxidoreductase Chromosomal Location of Human Ortholog: 15q26.1 Cellular Component: mitochondrion; mitochondrial matrix; mitochondrial inner membrane Molecular Function:magnesium ion binding; isocitrate dehydrogenase (NADP+) activity; NAD binding Biological Process: glyoxylate cycle; isocitrate metabolic process; cellular metabolic process; tricarboxylic acid cycle; carbohydrate metabolic process; 2-oxoglutarate metabolic process Disease: D-2-hydroxyglutaric Aciduria 2 |
| NCBI Summary: | Isocitrate dehydrogenases catalyze the oxidative decarboxylation of isocitrate to 2-oxoglutarate. These enzymes belong to two distinct subclasses, one of which utilizes NAD(+) as the electron acceptor and the other NADP(+). Five isocitrate dehydrogenases have been reported: three NAD(+)-dependent isocitrate dehydrogenases, which localize to the mitochondrial matrix, and two NADP(+)-dependent isocitrate dehydrogenases, one of which is mitochondrial and the other predominantly cytosolic. Each NADP(+)-dependent isozyme is a homodimer. The protein encoded by this gene is the NADP(+)-dependent isocitrate dehydrogenase found in the mitochondria. It plays a role in intermediary metabolism and energy production. This protein may tightly associate or interact with the pyruvate dehydrogenase complex. Alternative splicing results in multiple transcript variants. [provided by RefSeq, Feb 2014] |
| UniProt Code: | P48735 |
| NCBI GenInfo Identifier: | 20141568 |
| NCBI Gene ID: | 3418 |
| NCBI Accession: | P48735.2 |
| UniProt Secondary Accession: | P48735,Q96GT3, B2R6L6, B4DFL2, |
| UniProt Related Accession: | P48735 |
| Molecular Weight: | 452 |
| NCBI Full Name: | Isocitrate dehydrogenase |
| NCBI Synonym Full Names: | isocitrate dehydrogenase 2 (NADP+), mitochondrial |
| NCBI Official Symbol: | IDH2 |
| NCBI Official Synonym Symbols: | IDH; IDP; IDHM; IDPM; ICD-M; D2HGA2; mNADP-IDH |
| NCBI Protein Information: | isocitrate dehydrogenase [NADP], mitochondrial; isocitrate dehydrogenase [NADP], mitochondrial; NADP(+)-specific ICDH; oxalosuccinate decarboxylase |
| UniProt Protein Name: | Isocitrate dehydrogenase [NADP], mitochondrial |
| UniProt Synonym Protein Names: | ICD-M; IDP; NADP(+)-specific ICDH; Oxalosuccinate decarboxylase |
| Protein Family: | Isocitrate dehydrogenase |
| UniProt Gene Name: | IDH2 |
| UniProt Entry Name: | IDHP_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. |
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