|Detection Range:||0.31-20 ng/mL|
|Sample Volume Required Per Well:||100µL|
|Sample Type:||Serum, plasma and other biological fluids|
|Specificity:||This kit recognizes Mouse PDK4 in samples. No significant cross-reactivity or interference between Mouse PDK4 and analogues was observed.|
This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Mouse PDK4. Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Mouse PDK4 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Mouse PDK4, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Mouse PDK4. The concentration of Mouse PDK4 in samples can be calculated by comparing the OD of the samples to the standard curve.
|UniProt Protein Function:||PDHK4: 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. PDHK4 is abundant in heart, skeletal muscle, kidney, and pancreatic islets. 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. 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.|
|UniProt Protein Details:|
Protein type:Protein kinase, atypical; EC 2. 7. 11. 2; Kinase, protein; Mitochondrial; ATYPICAL group; PDHK family
Cellular Component: mitochondrial inner membrane; mitochondrion
Molecular Function:ATP binding; protein kinase activity; protein serine/threonine/tyrosine kinase activity; pyruvate dehydrogenase (acetyl-transferring) kinase activity
Biological Process: cellular response to starvation; glucose homeostasis; insulin receptor signaling pathway; positive regulation of defense response to virus by host; protein amino acid phosphorylation; regulation of acetyl-CoA biosynthetic process from pyruvate; regulation of bone resorption; regulation of fatty acid biosynthetic process; regulation of fatty acid oxidation; regulation of pH; response to starvation
|NCBI GenInfo Identifier:||12585303|
|NCBI Gene ID:||27273|
|NCBI Accession:||O70571. 1|
|UniProt Related Accession:||O70571|
|Molecular Weight:||46,596 Da|
|NCBI Full Name:||[Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial|
|NCBI Synonym Full Names:||pyruvate dehydrogenase kinase, isoenzyme 4|
|NCBI Official Symbol:||Pdk4|
|NCBI Official Synonym Symbols:||AV005916|
|NCBI Protein Information:||[Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial|
|UniProt Protein Name:||[Pyruvate dehydrogenase (acetyl-transferring)] kinase isozyme 4, mitochondrial|
|UniProt Synonym Protein Names:||Pyruvate dehydrogenase kinase isoform 4|
|UniProt Gene Name:||Pdk4|
|UniProt Entry Name:||PDK4_MOUSE|
As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e. g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.
Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse PDK4 were tested 20 times on one plate, respectively.
Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse PDK4 were tested on 3 different plates, 20 replicates in each plate.
|Intra-assay Precision||Inter-assay Precision|
|C V (%)||6.54||5.75||5.24||6.67||5.59||4.35|
The recovery of Mouse PDK4 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.
|Sample Type||Range (%)||Average Recovery (%)|
|EDTA plasma (n=5)||86-102||93|
|Cell culture media (n=5)||84-96||91|
Samples were spiked with high concentrations of Mouse PDK4 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.
|Serum (n=5)||EDTA plasma (n=5)||Cell culture media (n=5)|
An unopened kit can be stored at 4°C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.
|Micro ELISA Plate(Dismountable)||8 wells ×12 strips||-20°C, 6 months|
|Reference Standard||2 vials|
|Concentrated Biotinylated Detection Ab (100×)||1 vial, 120 µL|
|Concentrated HRP Conjugate (100×)||1 vial, 120 µL||-20°C(shading light), 6 months|
|Reference Standard & Sample Diluent||1 vial, 20 mL||4°C, 6 months|
|Biotinylated Detection Ab Diluent||1 vial, 14 mL|
|HRP Conjugate Diluent||1 vial, 14 mL|
|Concentrated Wash Buffer (25×)||1 vial, 30 mL|
|Substrate Reagent||1 vial, 10 mL||4°C(shading light)|
|Stop Solution||1 vial, 10 mL||4°C|
|Plate Sealer||5 pieces|
|Product Description||1 copy|
|Certificate of Analysis||1 copy|
- Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
- Aliquot 100µl of standard solutions into the standard wells.
- Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
- Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
- Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
- Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix. Incubate for 1 hour at 37°C.
- Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
- Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
- Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
- Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
- Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
- Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.