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Alias names |
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pyruvate dehydrogenase complex
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Specificity |
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High-throughput analysis of PDH E1α Ser 232, 293 and 300 phosphorylation in adherent cells
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Protocol |
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In-Cell ELISA Assay Kits use quantitative immunocytochemistry to measure protein levels or post-translational modifications in cultured cells. Cells are fixed in a 96- or 384-well plate and targets of interest are detected with highly specific, well-characterized monoclonal antibodies, and levels are quantified with IRDye®-labeled Secondary Antibodies. IR imaging and quantitation is performed using a LI-COR® Odyssey® or Aerius® system
For measuring the phosphorylation of PDH at all three of the enzyme's phospho-serine residues in adherent human, rat, mouse and bovine cells. This kit utilizes IRDyes® for detection, and so requires a LI-COR® Odyssey® or Aerius® imaging system. An alternate colorimetric version of this kit is available for use with standard plate readers - PhosphoPDH In-Cell ELISA Kit (Colorimetric) (catalog # MSP48).
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Storage |
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Store all components at 4°C. The kit is stable for at least 6 months
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Notes |
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MSP47 provides a high-throughput measurement of the amount of PDH E1α subunit in a cell and also the phosphorylation state of that protein subunit in a 96-well culture plate. To do this, the phosphorylation of E1α serine residues 232, 293, 300 is measured in parallel, while the total E1α is simultaneously measured in every well. The method uses In-cell ELISA (ICE) technology to perform this quantitative immunocytochemistry of cultured cells with near-infrared fluorescent dye-labeled detector antibodies. The technique generates quantitative data with specificity similar to Western blotting, but with much greater quantitative precision and higher throughput due to the greater dynamic range and linearity of direct fluorescence detection and the ability to run 96 samples in parallel. Since this method measures the total and phosphorylated E1α simultaneously in every well, phosphorylation can be normalized to the total level of E1α further increasing precision and throughput. This method rapidly fixes the cells in situ, stabilizing the phosphorylation state of the enzyme. This method essentially takes a snap-shot of phosphorylation state and rapid phosphorylation changes during sample handling are eliminated - a significant problem experienced during cell harvesting for other assays. Finally, both signals can also be normalized to a relative cell counting method (Janus Green cell stain1) if desired.
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