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Product name
PhosphoPDH In-Cell ELISA Kit (IR)
See all phosphoPDH products (2) ...
Tests
2 x 96 test
Sample type
Adherent cells
Assay type
Direct
Cross reactivity
Reacts with
Mouse, Rat, Cow, Human
Product overview
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.
Tested applications
In-Cell ELISAmore details
Properties
Storage instructions
Store at 4°C for upto 6 months. Refer to the protocol for further storage instructions.
| Components | 2 x 96 tests |
|---|---|
| 1000X IRDye-labeled Secondary Antibodies | 1 x 24µl |
| 100X Triton X-100 | 1 x 0.5ml |
| 10X Blocking Solution | 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 |
Research areas
Metabolism >> Pathways and Processes >> Metabolic signaling pathways >> Energy transfer pathways >> Energy Metabolism
Metabolism >> Pathways and Processes >> Metabolic signaling pathways >> Carbohydrate metabolism
Metabolism >> Pathways and Processes >> Mitochondrial Metabolism >> Mitochondrial markers
Kits/ Lysates/ Other >> Kits >> ELISA and ELIPAIR Kits >> ELISA Kits >> Growth factors and hormones ELISA kits
Kits/ Lysates/ Other >> Kits >> ELISA and ELIPAIR Kits >> ELISA Kits >> Cytokines and cytokine receptors ELISA kits
Cancer >> Cancer Metabolism >> Metabolic signaling pathway >> Metabolism of carbohydrates
Kits/ Lysates/ Other >> Kits >> Cell Metabolism Kits >> Other Metabolism Assay
Signal Transduction >> Metabolism >> Mitochondrial
Signal Transduction >> Metabolism >> Energy Metabolism
Applications
Show applications key
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
PhosphoPDH In-Cell ELISA Kit (IR) images:
In-Cell ELISA - PhosphoPDH In-Cell ELISA Kit (IR) (ab110218)
Cells (HepG2) were seeded at 50,000 cells per well and treated with millimolar concentrations of DCA in vehicle (1% DMSO) for 2 hours. Cells were then fixed and processed according to the protocol. To account for any cell density variability between wells, the ratio of PDH E1α pSer : total E1α signal is determined.
In-Cell ELISA - PhosphoPDH In-Cell ELISA Kit (IR) (ab110218)
Determination of the IC50 of dichloracetate in HepG2, HeLa and HDFn cells. The ratio of PDH E1α pSer232, 293 and 300 to the total E1α signal was normalized to the untreated (DMSO) sample for each concentration of DCA from 0-40 mM, showing that DCA is effective at inhibiting the PDH kinases and reducing phosphorylation at all three regulatory serine residues.
Other - PhosphoPDH In-Cell ELISA Kit (IR) (ab110218)
Antibody specificity demonstrated by Western Blot. HepG2 cells were treated with 5 mM dichloroacetate (DCA) for 4 hours to inhibit PDH kinase activity. To maintain the PDH in the maximal dephosphorylated state the cells were harvested quickly and washed in DCA containing buffers before Western blot analysis. Shown the PDH E1α and pSer293 analysis with antibodies used in this kit.
Immunocytochemistry - PhosphoPDH In-Cell ELISA Kit (IR) (ab110218)
Antibody specificity demonstrated by immunocytochemistry. Two-color immunocytochemical labeling of cultured HepG2 cells with the MSP47 primary antibodies specific for E1α PhosphoSer232, 293 and 300 and Total E1α. When the images are merged the two antibodies exhibit specific co-localization in the mitochondria.
In-Cell ELISA - PhosphoPDH In-Cell ELISA Kit (IR) (ab110218)
After cells have been grown to approximately 80% confluency in a 96- or 384-well plate, a drug or other treatment is applied to stimulate a cellular response. After treatment the cells are fixed and permeabilized in the wells, effectively "freezing" the cells in context with no further sample prep perturbations. Primary antibodies are then added which bind to their intended targets within the mitochondria or other subcellular compartment. After incubation, the unbound primary antibodies are washed away and secondary antibodies are added. These secondaries are conjugated to either IRDyes® or to an enzyme label (HRP or AP) for the colorimetric versions of the assays. Unbound secondaries are washed away, reaction buffer is added for the colorimetric assays, and the signal is read on a suitable instrument for the kit type.
» In-cell ELISA diagram in PDF format
Protocols
References for PhosphoPDH In-Cell ELISA Kit (IR) (ab110218)
ab110218 has not yet been referenced specifically in any publications.
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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"