Rat [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial (Pdk1) ELISA Kit
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![Rat [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial (Pdk1) ELISA Kit](https://cdn11.bigcommerce.com/s-d5atsu6k01/images/stencil/608x608/products/109661/109503/rat-pyruvate-dehydrogenase-lipoamide-kinase-isozyme-1-mitochondrial-pdk1-elisa-kit__80942__40292.1648049372.jpg?c=1 "Rat [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial (Pdk1) ELISA Kit")
- SKU:
- RTEB0606
- Product Type:
- ELISA Kit
- Size:
- 96 Assays
- Uniprot:
- Q63065
- Range:
- 0.156-10 ng/mL
- ELISA Type:
- Sandwich
- Synonyms:
- Pdk1, PDK1, PDPK1
- Reactivity:
- Rat
![Rat [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial (Pdk1) ELISA Kit](https://cdn11.bigcommerce.com/s-d5atsu6k01/images/stencil/1280x1280/products/109661/109503/rat-pyruvate-dehydrogenase-lipoamide-kinase-isozyme-1-mitochondrial-pdk1-elisa-kit__80942__40292.1648049372.jpg?c=1&imbypass=on "Rat [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial (Pdk1) ELISA Kit")
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Description
| 商品名: | Rat [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial (Pdk1) ELISA Kit |
| 製品コード: | RTEB0606 |
| エイリアス: | [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial, PDK p48, Pyruvate dehydrogenase kinase isoform 1, Pdk1, Pdh, 2.7.11.2, PDH kinase 1 |
| Uniprot: | Q63065 |
| 反応性: | Rat |
| 範囲: | 0.156-10 ng/mL |
| 検出方法: | Sandwich |
| サイズ: | 96 Assay |
| 保管所: | Please see kit components below for exact storage details |
| ノート: | For research use only |
| UniProt Protein Function: | PDHK1: an ubiquitously expressed, atypical protein kinase associated with the mitochondrial matrix. The PDHKs play crucial roles in switching metabolic flux from oxidative phosphorylation towards glycolysis. PDHK1 is detected in heart, pancreatic islets, and skeletal muscles. Contains a HATPase_c catalytic domain, found in several ATP-binding proteins including protein histidine kinases (PHKs), PHDKs, DNA gyrase B, topoisomerases, heat shock proteins, and DNA mismatch repair proteins. PDHK regulates glucose oxidation through inhibitory phosphorylation of the E1 alpha subunit of the mitochondrial pyruvate dehydrogenase complex (PDHC) at any one of 3 inhibitory serine residues. Inhibitory sites 1, 2, and 3 correspond to S293, S300, and S232 in human PDHA1, respectively. Four PDHK isoenzymes have been described, each with different site specificity: all four phosphorylate sites 1 and 2 but at different rates; for site 1 PDHK2 >PDHK4 >PDHK1 >PDHK3; for site 2, PDHK3> PDHK4 > PDHK2 > PDHK1. Only PDHK1 phosphorylates site 3. PDHKs are recruited to the PDHC by binding to a lipoyl group covalently attached to the inner lipoyl domain of the E2 component. PDHA1 deficiency is the most common enzyme defect in patients with primary lactic acidosis. PDHK1 is a direct HIF-1 target gene. Suppression of PDH by PDHK inhibits the conversion of pyruvate to acetyl-CoA, attenuates mitochondrial respiration, and may contribute to the increased lactate production observed in many tumors. The PDH pathway is repressed in a majority of non-small cell lung carcinomas. Inhibited by AZD7545, dichloroacetate (DCA) and radicicol. Radicicol inhibits kinase activity by binding directly to the ATP-binding pocket of PDHK, similar to HSP90 from the same ATPase/kinase superfamily.Protein type: ATYPICAL group; EC 2.7.11.2; Kinase, protein; Mitochondrial; PDHK family; Protein kinase, atypicalChromosomal Location of Human Ortholog: 3q23Cellular Component: mitochondrial pyruvate dehydrogenase complex; mitochondrion; nucleolus; nucleus; pyruvate dehydrogenase complexMolecular Function: ATP binding; protein complex binding; protein heterodimerization activity; protein homodimerization activity; protein kinase activity; protein serine/threonine kinase activity; pyruvate dehydrogenase (acetyl-transferring) kinase activityBiological Process: cell proliferation; cell surface receptor linked signal transduction; induction of apoptosis by oxidative stress; peptidyl-serine phosphorylation; protein amino acid phosphorylation; regulation of acetyl-CoA biosynthetic process from pyruvate |
| UniProt Protein Details: | |
| NCBI Summary: | catalyzes the phosphorylation and inactivation of the pyruvate dehydrogenase complex [RGD, Feb 2006] |
| UniProt Code: | Q63065 |
| NCBI GenInfo Identifier: | 3183109 |
| NCBI Gene ID: | 116551 |
| NCBI Accession: | Q63065.1 |
| UniProt Secondary Accession: | Q63065 |
| UniProt Related Accession: | Q63065 |
| Molecular Weight: | 49,081 Da |
| NCBI Full Name: | [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1, mitochondrial |
| NCBI Synonym Full Names: | pyruvate dehydrogenase kinase 1 |
| NCBI Official Symbol: | Pdk1 |
| NCBI Official Synonym Symbols: | |
| NCBI Protein Information: | pyruvate dehydrogenase kinase, isozyme 1 |
| UniProt Protein Name: | [Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 1, mitochondrial |
| UniProt Synonym Protein Names: | PDK p48; Pyruvate dehydrogenase kinase isoform 1; PDH kinase 1 |
| Protein Family: | [Pyruvate dehydrogenase |
| UniProt Gene Name: | Pdk1 |
| UniProt Entry Name: |
| 成分 | 額 | 保管所 |
| 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 | - |
その他の必要な材料と設備：
- 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
*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。
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.
| ステップ | プロトコル |
| 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. |
ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。
| サンプルタイプ: | プロトコル: |
| 血清: | 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. |
| プラズマ: | 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. |
| 尿および脳脊髄液: | 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. |
| 細胞培養上清: | 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. |
| 細胞溶解物: | 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. |
| 組織ホモジネート: | 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. |
| 組織溶解物: | 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. |
| 母乳: | 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. |