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|Sample type||Average %||Range|
|Serum||144||129% - 152%|
|Cell culture media||135||134% - 137%|
|Fetal Bovine Serum||135||129% - 130%|
Abcam’s PDH E1 alpha protein in vitro Human SimpleStep ELISA™ (Enzyme-Linked Immunosorbent Assay) kit is designed for the accurate quantitative measurement of PDH E1 alpha protein in Human cell and tissue extracts.
The SimpleStep ELISA™ employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm.Optionally,instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
The pyruvate dehydrogenase complex performs the decarboxylation of pyruvate into acetyl CoA, a critical function in metabolism, linking glycolysis and oxidative phosphorylation in mitochondria. The enzyme is composed of multiple copies of three enzymes: pyruvate dehydrogenase (E1), dihydrolipoamide transacetylase (E2) and dihydrolipoamide dehydrogenase (E3). The E1 enzyme is a tetramer of two alpha (PDHA1) and two beta (PDHB) subunits and is present in 30 copies in the PDH complex.
The activity of PDH is regulated by reversible phosphorylation of three serine residues on the PDHA1 subunit at phospho S232, phospho S293, and phospho S300. ELISA assays to measure total PDHA1, phospho 232, phospho S293, and phospho S300 are available from Abcam (ab115342, ab115343, ab115344, ab115345). The phosphorylation of these sites is catalyzed by PDH kinases (PDK). There are four known PDK isoforms, distributed differently in tissues. Their expressions are regulated differently by factors such as starvation, hypoxia and utilization of glucose and fatty acids in various tissues. Dephosporylation, to restore the activity of PDH, is catalyzed by PDH phosphatases (PDP). There are two known isoforms of PDP; PDP1 is present in high levels in skeletal muscle and PDP2 in liver and adipocytes. Functional PDH kinases and phosphatases are available from Abcam (ab110359, ab110354, ab110355, ab110356).
|Components||1 x 96 tests|
|10X PDH E1a Capture Antibody||1 x 600µl|
|10X PDH E1a Detector Antibody||1 x 600µl|
|10X Wash Buffer LM||1 x 20ml|
|2X Cell Extraction Buffer LM||1 x 10ml|
|4X Antibody Diluent EB||1 x 6ml|
|PDH E1 alpha Human Lyophilized Recombinant Protein||2 vials|
|Plate Seals||1 unit|
|Pre-Coated 96-Well Microplate||1 unit|
|Sample Diluent NS||1 x 12ml|
|Stop Solution||1 x 12ml|
|TMB Substrate||1 x 12ml|
Our Abpromise guarantee covers the use of ab181415 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|Sandwich ELISA||Use at an assay dependent concentration.|
Example of PDH E1 alpha (PDHA1) standard curve. Background-subtracted data values (mean +/- SD) are graphed.
Titration of HeLa, HepG2, Human Liver Homogenate (HLH) and MCF7 cell extract within the working range of the assay in 1X Cell Extraction Buffer LM. Background subtracted data from duplicate measurements are plotted.
ab181415 has not yet been referenced specifically in any publications.