Pdk4 ELISA Kit (Mouse)

Intra-assay Precision (Precision within an assay) : 3 samples with low, middle and high level Pyruvate Dehydrogenase Kinase 4 (PDK4) were tested 20 times on one plate, respectively. Inter-assay Precision (Precision between assays) : 3 samples with low, middle and high level Pyruvate Dehydrogenase Kinase 4 (PDK4) were tested on 3 different plates, 8 replicates in each plate. CV (%) = SD/meanX100 Intra-Assay: CV<10% Inter-Assay: CV<12%

[Pyruvate dehydrogenase (acetyl-transferring) ] kinase isozyme 4, mitochondrial; [Pyruvate dehydrogenase [lipoamide]] kinase isozyme 4, mitochondrial; AV005916; pyruvate dehydrogenase kinase 4; Pyruvate dehydrogenase kinase isoform 4.

Kinase that plays a key role in regulation of glucose and fatty acid metabolism and homeostasis via phosphorylation of the pyruvate dehydrogenase subunits PDHA1 and PDHA2. This inhibits pyruvate dehydrogenase activity, and thereby regulates metabolite flux through the tricarboxylic acid cycle, down-regulates aerobic respiration and inhibits the formation of acetyl-coenzyme A from pyruvate. Inhibition of pyruvate dehydrogenase decreases glucose utilization and increases fat metabolism in response to prolonged fasting and starvation. Plays an important role in maintaining normal blood glucose levels under starvation, and is involved in the insulin signaling cascade. Via its regulation of pyruvate dehydrogenase activity, plays an important role in maintaining normal blood pH and in preventing the accumulation of ketone bodies under starvation. In the fed state, mediates cellular responses to glucose levels and to a high-fat diet. Regulates both fatty acid oxidation and de novo fatty acid biosynthesis. Plays a role in the generation of reactive oxygen species. Protects detached epithelial cells against anoikis. Plays a role in cell proliferation via its role in regulating carbohydrate and fatty acid metabolism.

The test principle applied in this kit is Sandwich enzyme immunoassay. The microtiter plate provided in this kit has been pre-coated with an antibody specific to Pyruvate Dehydrogenase Kinase 4 (PDK4) . Standards or samples are then added to the appropriate microtiter plate wells with a biotin-conjugated antibody specific to Pyruvate Dehydrogenase Kinase 4 (PDK4) . Next, Avidin conjugated to Horseradish Peroxidase (HRP) is added to each microplate well and incubated. After TMB substrate solution is added, only those wells that contain Pyruvate Dehydrogenase Kinase 4 (PDK4), biotin-conjugated antibody and enzyme-conjugated Avidin will exhibit a change in color. The enzyme-substrate reaction is terminated by the addition of sulphuric acid solution and the color change is measured spectrophotometrically at a wavelength of 450nm +/- 10nm. The concentration of Pyruvate Dehydrogenase Kinase 4 (PDK4) in the samples is then determined by comparing the O.D. of the samples to the standard curve.

Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Reconstitution & Storage Instructions Reconstitution & Storage Instructions Western Blotting/Immunoblotting (WB/IB) Protocol Western Blotting/Immunoblotting (WB/IB) Protocol Immunohistochemistry (IHC) Protocol Immunohistochemistry (IHC) Protocol Immunocytochemistry (ICC) Protocol Immunocytochemistry (ICC) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Enzyme-Linked ImmunoSorbent Assay (ELISA) Protocol Blocking Peptide Competition Protocol (BPCP) Blocking Peptide Competition Protocol (BPCP) Immunoprecipitation (IP) Protocol Immunoprecipitation (IP) Protocol Antibody Array (AA) Protocol Antibody Array (AA) Protocol

Matrix Recovery Range (%) Average (%) Serum (n=5) 92-99 95 EDTA Plasma (n=5) 88-96 93 Heparin Plasma (n=5) 90-97 94 Matrix Recovery Range (%) Average (%) Matrix Recovery Range (%) Average (%) Serum (n=5) 92-99 95 Serum (n=5) 92-99 95 EDTA Plasma (n=5) 88-96 93 EDTA Plasma (n=5) 88-96 93 Heparin Plasma (n=5) 90-97 94 Heparin Plasma (n=5) 90-97 94

Component Amount Anti-Pdk4 Microplate 96 Wells (12 x 8 Well Strips) Pdk4 Lyophilized Standard 2 x 20 ng 100X Biotinylated Pdk4 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Component Amount Component Amount Anti-Pdk4 Microplate 96 Wells (12 x 8 Well Strips) Anti-Pdk4 Microplate 96 Wells (12 x 8 Well Strips) Pdk4 Lyophilized Standard 2 x 20 ng Pdk4 Lyophilized Standard 2 x 20 ng 100X Biotinylated Pdk4 Detector Antibody 120 uL 100X Biotinylated Pdk4 Detector Antibody 120 uL Avidin/HRP Conjugate 120 uL Avidin/HRP Conjugate 120 uL Standard Diluent 1 x 20 mL Standard Diluent 1 x 20 mL Detector Antibody Diluent 1 x 12 mL Detector Antibody Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL Conjugate Diluent 1 x 12 mL 30X Wash Buffer 1 x 20 mL 30X Wash Buffer 1 x 20 mL TMB Substrate 1 x 9 mL TMB Substrate 1 x 9 mL Stop Solution 1 x 6 mL Stop Solution 1 x 6 mL

This assay has high sensitivity and excellent specificity for detection of Pyruvate Dehydrogenase Kinase Isozyme 4 (PDK4) . No significant cross-reactivity or interference between Pyruvate Dehydrogenase Kinase Isozyme 4 (PDK4) and analogues was observed.