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In-Cell ELISA 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.
ab110218(MSP47) is a high-throughput assay for measuring up- or down-regulation of total pyruvate dehydrogenase (PDH) subunit E1a, as well as phosphorylation at all three E1a regulatory serines: Ser232, Ser293 and Ser300. The assay is designed for use with small numbers of cultured adherent cells in a 96-well microplate but can easily be adapted to a 384-well plate.
ab110218 provides a high-throughput measurement of the amount of PDH E1a 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 E1a serine residues 232, 293, 300 is measured in parallel, while the total E1a is simultaneously measured in every well. The method uses In-Cell ELISA 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 E1a simultaneously in every well, phosphorylation can be normalized to the total level of E1a 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 stain) if desired.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
|组件||2 x 96 tests|
|1000X IRDye-labeled Secondary Antibodies||1 x 24µl|
|100X Triton X-100||1 x 0.5ml|
|10X Blocking Buffer||1 x 15ml|
|10X Phosphate Buffered Saline||1 x 100ml|
|1X Janus Green Stain||1 x 11ml|
|200X E1a pSer232 Primary Antibody||1 x 40µl|
|200X E1a pSer293 Primary Antibody||1 x 40µl|
|200X E1a pSer300 Primary Antibody||1 x 40µl|
|200X Total E1a Primary Antibody||1 x 0.11ml|
|400X Tween-20||1 x 2ml|
|Plate Seals||2 units|
Our Abpromise guarantee covers the use of ab110218 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|In-Cell ELISA||Use at an assay dependent dilution.|
ab110218 has not yet been referenced specifically in any publications.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"