ab115343 is an in vitro enzyme-linked immunosorbent assay to determine the levels of phospho S232 PDHA1 protein in cell and tissue lysates. The assay employs a mouse antibody specific for PDHA1 protein coated on a 96-well plate. Samples are pipetted into the wells and PDHA1 protein present in the sample is bound to the wells by the immobilized antibody. The wells are washed and a rabbit anti-phospho S232 PDHA1 protein detector antibody is added. After washing away unbound detector antibody, HRP-conjugated anti-rabbit antibody is pipetted into the wells. The wells are again washed, an HRP substrate solution (TMB) is added to the wells and color develops in proportion to the amount of phospho S232 PDHA1 protein bound. The developing blue color is measured at 600 nm. Optionally the reaction can be stopped by adding hydrochloric acid which changes the color from blue to yellow and the intensity can be measured at 450 nm.
Store all components at 4°C. The kits are stable for at least 6
months. Unused microplate strips should be returned to the pouch
containing the desiccant and resealed.
|Components||1 x 96 tests|
|10X Blocking Buffer||1 x 6ml|
|10X HRP Label||1 x 1ml|
|10X phospho S232 PDHA1 protein Detector Antibody||1 x 0.7ml|
|20X Buffer||1 x 20ml|
|Extraction Buffer||1 x 15ml|
|Microplate 96 antibody coated wells in 12 strips||1 unit|
|HRP Development Solution||1 x 12ml|
Our Abpromise guarantee covers the use of ab115343 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ELISA||Use at an assay dependent concentration.|
The PDHA1 bound from undosed HeLa cells was subject to in-well kinase treatment (PDK1&3) or in-well phosphatase treatment (PDP1) according to the supplementary protocol shown below. Untreated cells showed a significant endogenous phosphorylation signal at S232 which is only slightly increased by kinase treatment. Conversely phosphatase treatment was able to significantly reduce the phospho S232 signal from the endogenous levels.
Example control sample curve for HepG2 cell extract. The phosphorylation state of PDHA1 can vary by treatment but also by cell culture conditions such as media supplements, nutrients and also cell density.
Example control sample curve for HeLa cell extract. The phosphorylation state of PDHA1 can vary by treatment but also by cell culture conditions such as media supplements, nutrients and also cell density.
ab115343 has not yet been referenced specifically in any publications.