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|HeLa cells||< 5%|
ab125300 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure HIF1 alpha and Actin 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 reference protein actin.
HIF1 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 take longer to manifest. The anti-HIF1 alpha antibody used in this assay is specific for Human HIF1 alpha protein.
Actin is a ubiquitously expressed component of the cellular cytoskeleton and is involved in cell motility, cytokinesis and muscle contraction. Given its abundance and ubiquity, actin is a commonly used normalizing signal in cell biology experiments. The anti-Actin antibody used in this assay reacts with all six vertebrate isoforms of actin.
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 in the preparation of protein extracts. Finally, the HIF1 alpha and Actin 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 Actin Primary Antibody (Mouse)||1 x 120µl|
|100X HIF1alpha Primary Antibody (Rabbit)||1 x 120µl|
|100X Triton X-100||1 x 0.5ml|
|10X Blocking Buffer||1 x 10ml|
|10X Phosphate Buffered Saline||1 x 100ml|
|400X Tween-20||1 x 2ml|
|Janus Green Stain||1 x 17ml|
Our Abpromise guarantee covers the use of ab125300 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.|
ab125300 has not yet been referenced specifically in any publications.