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|HeLa cells||< 8%|
ab125299 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure HIF1 alpha and PDK1 protein levels in cultured cells. Cells are fixed in a microplate and targets of interest are detected with highly specific, well-characterized antibodies. Relative protein levels are quantified using IRDye®-labeled Secondary Antibodies and IR imaging using a LI-COR® Odyssey® or Aerius® system.
Hypoxia and the cellular response to hypoxic environment is a central topic in studies of metabolism, cancer progression and development and stem cells. A key player is the transcription factor HIF1 alpha (hypoxia inducible factor 1 alpha) which is stabilized at the protein level in response to decreased oxygen tension. HIF1 alpha then promotes transcription of a number of factors that alters cellular physiology. This Hypoxia ICE assay kit provides duplexed measurements of HIF1 alpha and the HIF1 alpha responsive proteins PDK1.
HIF1 alpha (hypoxia-inducible factor 1-alpha) is a constitutively expressed transcription factor that is degraded under normal oxygen tensions but stabilized (at the protein level) when oxygen is limiting (hypoxia). Under hypoxic conditions, stabilized HIF1A promotes the transcription of a host of genes that enable the cell to adapt to the lack of oxygen. A key aspect of the hypoxic response is the switch from aerobic respiration to anaerobic glycolysis and many of the HIF1 alpha responsive genes encode proteins that promote glycolysis and/or inhibit oxidative phosphorylation. Stabilization of the HIF1 alpha protein levels can be detected quickly after the onset of hypoxia, whereas changes in the levels of HIF1 alpha responsive proteins takes longer to manifest. The anti-HIF1 alpha antibody used in this assay is specific for Human HIF1 alpha protein.
PDK1 (dehydrogenase kinase isozyme 1, mitochondrial) is a mitochondrial matrix protein that contributes to the regulation of glucose metabolism. PDK1 inhibits the mitochondrial pyruvate dehydrogenase complex (PDH) by phosphorylation of the E1 alpha subunit. The pyruvate dehydrogenase complex transforms pyruvate to Acetyl-CoA for use in the TCA cycle. Hence, inhibitory phosphorylation events by PDK1 on PDH function to down-regulate TCA and oxidative phosphorylation. Stabilization of the HIF1 alpha transcription factor directly leads to increased transcription of PDK1. The PDK1 antibody used in this assay reacts with Human and Bovine PDK1.
In-Cell ELISA (ICE) technology is used to perform quantitative immunocytochemistry of cultured cells with a near-infrared fluorescent dye-labeled detector antibody. 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. This method rapidly fixes the cells in situ, stabilizing the in vivo levels of proteins, and thus essentially eliminates changes during sample handling, such as preparation of protein extracts. Finally, the HIF1 alpha and PDK1 signals can all be normalized to cell amount, measured by the provided Janus Green whole cell stain, to further increase the assay precision.
Plates are available in our ICE (In-Cell ELISA) Support Pack (ab111542) which can be bought seperately.
Upon receipt spin down the contents of the IRDye®-labeled Secondary Antibody tube and protect from light. Store all components upright at 4°C. This kit is stable for at least 6 months from receipt.
|Components||1 x 96 tests|
|1000X IRDye-labeled Secondary Antibodies||1 x 24µl|
|100X HIF1alpha Primary Antibody (Rabbit)||1 x 120µl|
|100X PDK1 Primary Antibody (Mouse)||1 x 120µl|
|100X Triton X-100||1 x 1.5ml|
|10X Blocking Solution||1 x 15ml|
|10X Phosphate Buffered Saline||1 x 100ml|
|400X Tween-20||1 x 2ml|
|1X Janus Green Stain||1 x 17ml|
Our Abpromise guarantee covers the use of ab125299 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 concentration.|
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"