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|HeLa cells||< 8%|
ab125298 is an In-Cell ELISA (ICE) assay kit that uses quantitative immunocytochemistry to measure HIF1 alpha and GLUT1 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 the transcription factor HIF1 alpha and the HIF1A responsive protein GLUT1.
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.
GLUT1 is a widely expressed cell membrane glucose transporter and is responsible for basal glucose uptake. GLUT1 can transport a range of aldolases including pentoses and hexoses. Stabilization of the HIF1 alpha transcription factor directly leads to increased transcription of GLUT1. In turn, increased expression of GLUT1 in response to hypoxia is thought to provide additional sugars for anaerobic glycolysis. The gene name for GLUT1 is Solute carrier family 2, facilitated glucose transporter member 1 (SLC2A1). The anti-GLUT1 antibody in this assay kit is reactive with Human, Mouse and Rat GLUT1.
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 GLUT1 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.
|内容||1 x 96 tests|
|1000X IRDye-labeled Secondary Antibodies||1 x 24µl|
|100X GLUT1 Primary Antibody (Rabbit)||1 x 120µl|
|100X HIF1alpha Primary Antibody (Mouse)||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 ab125298 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.|
ab125298 has not yet been referenced specifically in any publications.