Human Hif1 + PDK1 Hypoxia In Cell ELISA Kit (IR) (ab125299)

製品の概要

  • 製品名Human Hif1 + PDK1 Hypoxia In Cell ELISA Kit (IR)
  • 検出方法IR
  • 再現性
    Intra-Assay(同時再現性)
    サンプル N 平均値 SD CV%
    HeLa cells < 8%
  • テスト
    1 x 96 test
  • サンプルの種類
    Adherent cells, Suspension cells
  • アッセイタイプCell-based (quantitative)
  • ステップMultiple steps standard assay
  • 種交差性
    交差種: Human
  • 製品の概要

    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.

     

  • 特記事項

    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.

  • アプリケーション適用あり: In-Cell ELISAmore details
  • 試験プラットフォームMicroplate

製品の特性

  • 細胞内局在Mitochondrial and Nuclear
  • 別名
    • HBP231
    • HIF-1
    • HIP-1
    • Histone-lysine N-methyltransferase SETD2
    • HSPC069
    • Huntingtin yeast partner B
    • Huntingtin-interacting protein 1
    • Huntingtin-interacting protein B
    • HYPB
    • KMT3A
    • Lysine N-methyltransferase 3A
    • MS1008
    • p231HBP
    • Pyruvate dehydrogenase [lipoamide]] kinase isozyme 1, mitochondrial
    • Pyruvate dehydrogenase kinase isoform 1
    • Pyruvate dehydrogenase kinase, isozyme 1
    • SET domain containing 2
    • SET domain-containing protein 2
    • SET2
    see all
  • 参照データベース

アプリケーション

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.

アプリケーション Abreviews 特記事項
In-Cell ELISA Use at an assay dependent concentration.

Human Hif1 + PDK1 Hypoxia In Cell ELISA Kit (IR) 画像

  • Sample experiment using ab125299 on HeLa cells treated with a titration of DFO. HeLa cells were seeded to an amine coated 96-well microplate and the following day treated with a titration of DFO. After 24h of DFO exposure, the cells were fixed and stained as described in the protocol and the normalized data is presented here +/- SD (as described in the protocol and data analysis sections). HIF1 alpha results show DFO concentrations >10µM induce HIF1A protein levels in a dose dependent fashion.
  • Sample experiment using ab125299 on HeLa cells treated with a titration of DFO. HeLa cells were seeded to an amine coated 96-well microplate and the following day treated with a titration of DFO. After 24h of DFO exposure, the cells were fixed and stained as described in the protocol and the normalized data is presented here +/- SD (as described in the protocol and data analysis sections). PDK1 levels are increased with >10µM DFO.
  • Antibody specificity demonstrated by immunocytochemistry. Primary antibodies used in this assay kit were validated by staining HeLa cells +/- treatment with 1mM DFO (24h) and imaged by fluorescent microscopy. HIF1 alpha (ab51608, diluted 1:2000) staining is absent in untreated cells and induced by DFO treatment. HIF1 alpha localizes to the nucleus (as seen by co-localization with the DNA stain DAPI) as expected.
  • Antibody specificity demonstrated by immunocytochemistry. Primary antibodies used in this assay kit were validated by staining HeLa cells +/- treatment with 1mM DFO (24h) and imaged by fluorescent microscopy. PDK1 staining (ab110335, diluted 2µg/mL) labels cellular mitochondria and the fluorescent intensity increases with DFO treatment.
  • Antibody specificity demonstrated by western blot. Primary antibodies used in this assay kit were validated by western blot using HeLa cell lysates that had been treated with a dose titration of DFO as indicated. (A) The HIF1 alpha (ab51608) band (indicated by arrow) is absent in untreated cells and induced in a dose-dependent manner by DFO. (B) Similarly, PDK1 levels are increased by DFO treatment in a dose-dependent manner.
  • Coefficient of variation for the experiment described in Figure 1.

プロトコール

Human Hif1 + PDK1 Hypoxia In Cell ELISA Kit (IR) (ab125299) 使用論文

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Product Wall

1. The amine coated plates that we use are from BD Biosciences
BD PureCoat™ Microplates
black/clear, polystyrene
96-well, amine 5
cat # 354717
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Thank you for your inquiry. Unfortunately, you must have a LI-COR-Odyssey or Aerius system to use this kit. Measurement of Hif1a and PDK1 is done using IR dye conjugated secondary antibodies which need to be quantified using these systems.

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The IRDye labeled secondary antibodies provided in this kit are a mixture of IRDye 800 and IRDye 680 (standard Odyssey IR dyes that are read on the Odyssey scanner in the 800 and 700 channels). Section 8.11 in the protocol booklet. From LICOR’s w...

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Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"