Description
Human Dihydropteridine reductase (QDPR) ELISA Kit
The Human Dihydropteridine Reductase (QDPR) ELISA Kit is specifically designed for the precise measurement of QDPR levels in human samples such as serum, plasma, and cell culture supernatants. With its high sensitivity and specificity, this kit can provide dependable and consistent results, making it an excellent tool for various research applications.QDPR is an essential enzyme involved in the metabolism of tetrahydrobiopterin (BH4), a cofactor necessary for the proper functioning of enzymes involved in neurotransmitter synthesis and nitric oxide production.
Dysregulation of QDPR activity has been associated with various genetic disorders, including hyperphenylalaninemia and dopa-responsive dystonia.By accurately measuring QDPR levels, researchers can gain valuable insights into the mechanisms underlying these disorders and potentially identify new therapeutic targets. The Human Dihydropteridine Reductase (QDPR) ELISA Kit offers a reliable and efficient method for investigating QDPR levels in human samples, opening up new possibilities for advancing our understanding of these complex conditions.
| Product Name: | Human Dihydropteridine reductase (QDPR) ELISA Kit |
| SKU: | HUEB1600 |
| Size: | 96T |
| Target: | Human Dihydropteridine reductase (QDPR) |
| Synonyms: | HDHPR, Quinoid dihydropteridine reductase, Short chain dehydrogenase/reductase family 33C member 1, DHPR, SDR33C1 |
| Assay Type: | Sandwich |
| Detection Method: | ELISA |
| Reactivity: | Human |
| Detection Range: | 0.312-20ng/mL |
| Sensitivity: | 0.3ng/ml |
| Intra CV: | 4.1% | ||||||||||||||||||||
| Inter CV: | 6.5% | ||||||||||||||||||||
| Linearity: |
| ||||||||||||||||||||
| Recovery: |
| ||||||||||||||||||||
| Function: | The product of this enzyme, tetrahydrobiopterin (BH-4), is an essential cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases. |
| Uniprot: | P09417 |
| Sample Type: | Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids |
| Specificity: | Natural and recombinant human Dihydropteridine reductase |
| Sub Unit: | Homodimer. |
| Research Area: | Cell Biology |
| Storage: | Please see kit components below for exact storage details |
| Note: | For research use only |
| UniProt Protein Function: | QDPR: The product of this enzyme, tetrahydrobiopterin (BH-4), is an essential cofactor for phenylalanine, tyrosine, and tryptophan hydroxylases. Defects in QDPR are the cause of BH4-deficient hyperphenylalaninemia type C (HPABH4C); also called dihydropteridine reductase deficiency (DHPR deficiency) or hyperphenylalaninemia tetrahydrobiopterin-deficient due to DHPR deficiency or quinoid dihydropteridine reductase deficiency (QDPR deficiency). HPABH4C is a rare autosomal recessive disorder characterized by hyperphenylalaninemia and severe neurologic symptoms (malignant hyperphenylalaninemia) including axial hypotonia and truncal hypertonia, abnormal thermogenesis, and microcephaly. These signs are attributable to depletion of the neurotransmitters dopamine and serotonin, whose syntheses are controlled by tryptophan and tyrosine hydroxylases that use BH-4 as cofactor. These patients do not respond to phenylalanine- restricted diet. HPABH4C is lethal if untreated. Belongs to the short-chain dehydrogenases/reductases (SDR) family. |
| UniProt Protein Details: | Protein type:Oxidoreductase; Cofactor and Vitamin Metabolism - folate biosynthesis; EC 1.5.1.34 Chromosomal Location of Human Ortholog: 4p15.31 Cellular Component: cytoplasm; cytosol Molecular Function:6,7-dihydropteridine reductase activity; electron carrier activity Biological Process: amino acid metabolic process; dihydrobiopterin metabolic process; L-phenylalanine catabolic process Disease: Hyperphenylalaninemia, Bh4-deficient, C |
| NCBI Summary: | This gene encodes the enzyme dihydropteridine reductase, which catalyzes the NADH-mediated reduction of quinonoid dihydrobiopterin. This enzyme is an essential component of the pterin-dependent aromatic amino acid hydroxylating systems. Mutations in this gene resulting in QDPR deficiency include aberrant splicing, amino acid substitutions, insertions, or premature terminations. Dihydropteridine reductase deficiency presents as atypical phenylketonuria due to insufficient production of biopterin, a cofactor for phenylalanine hydroxylase. [provided by RefSeq, Jul 2008] |
| UniProt Code: | P09417 |
| NCBI GenInfo Identifier: | 118572639 |
| NCBI Gene ID: | 5860 |
| NCBI Accession: | P09417.2 |
| UniProt Secondary Accession: | P09417,Q53F52, Q9H3M5, A8K158, B3KW71, |
| UniProt Related Accession: | P09417 |
| Molecular Weight: | 22,408 Da |
| NCBI Full Name: | Dihydropteridine reductase |
| NCBI Synonym Full Names: | quinoid dihydropteridine reductase |
| NCBI Official Symbol: | QDPR |
| NCBI Official Synonym Symbols: | DHPR; PKU2; SDR33C1 |
| NCBI Protein Information: | dihydropteridine reductase |
| UniProt Protein Name: | Dihydropteridine reductase |
| UniProt Synonym Protein Names: | HDHPR; Quinoid dihydropteridine reductase; Short chain dehydrogenase/reductase family 33C member 1 |
| Protein Family: | Dihydropteridine reductase |
| UniProt Gene Name: | QDPR |
| UniProt Entry Name: | DHPR_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. |
