概述

  • 产品名称
    人HIF-1 alpha ELISA试剂盒
    参阅全部 HIF-1 alpha 试剂盒
  • 检测方法
    Colorimetric
  • 精确度
    批次内
    样品 n Mean SD CV%
    HeLa lysates 5 4.3%
    批次间
    样品 n Mean SD CV%
    HeLa lysates 3 7%
  • 样品类型
    Cell culture extracts
  • 检测类型
    Sandwich (quantitative)
  • 灵敏度
    42 pg/ml
  • 范围
    0.23 ng/ml - 15 ng/ml
  • 回收率

    特定样本回收率
    样品类型 平均% 范围
    Cell culture media 112 94% - 129%
    Fetal Bovine Serum 109 102% - 120%
    Bovine Serum Albumin 97 93% - 106%

  • 检测时间
    1h 30m
  • 实验步骤
    One step assay
  • 种属反应性
    与反应: Human
    不与反应: Mouse, Rat
  • 产品概述

    Abcam’s HIF1a in vitro SimpleStep ELISA® (Enzyme-Linked Immunosorbent Assay) kit is designed for the quantitative measurement of HIF1a protein in Human cell extracts.

    The SimpleStep ELISA® employs an affinity tag labeled capture antibody and a reporter conjugated detector antibody which immunocapture the sample analyte in solution. This entire complex (capture antibody/analyte/detector antibody) is in turn immobilized via immunoaffinity of an anti-tag antibody coating the well. To perform the assay, samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material. TMB substrate is added and during incubation is catalyzed by HRP, generating blue coloration. This reaction is then stopped by addition of Stop Solution completing any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.

  • 说明

    Hypoxia-inducible factor 1-alpha (HIF1 alpha) is a constitutively expressed transcription factor that is degraded under normal oxygen tensions but stabilized when oxygen is limiting (hypoxia). Under hypoxic conditions, stabilized HIF1 alpha translocates to the nucleus and promotes the transcription of a host of genes that enable the cell to adapt to the lack of oxygen. Aspects of the HIF1 alpha mediated hypoxic response include promotion of angiogenesis and the switch from aerobic respiration to anaerobic glycolysis. Many of the HIF1 alpha responsive genes encode proteins that promote glycolysis and/or inhibit oxidative phosphorylation (known as the Warburg effect). An exciting and developing area of current cancer research is examining how HIF-mediated metabolic reprogramming promotes tumor growth and survival.

    In most cases, HIF1 alpha will need to be stabilized to be measured (steady state levels of HIF1 alpha in non-hypoxic environments is exceeding low in most cell lines). This can be achieved by (a) creating a hypoxic environment (e.g. using a hypoxia chamber) or (b) by using chemical treatments that mimic hypoxia (e.g. cobalt chloride or deferoxamine). The sample data in this assay protocol was generated using deferoxamine (DFO). DFO is an iron chelator and disrupts the function the prolyl hydroxylases that degrade HIF1 alpha in normoxia. By disrupting the enzymes that degrade HIF1 alpha, DFO increases the abundance of HIF1 alpha protein.

  • 经测试应用
    适用于: Sandwich ELISAmore details
  • 平台
    Microplate

性能

  • 存放说明
    Store at +4°C. Please refer to protocols.
  • 组件 1 x 96 tests
    10X HIF1a Capture Antibody 1 x 600µl
    10X HIF1a Detector Antibody 1 x 600µl
    10X Wash Buffer PT (ab206977) 1 x 20ml
    50X Cell Extraction Enhancer Solution (ab193971) 1 x 1ml
    5X Cell Extraction Buffer PTR (ab193970) 1 x 10ml
    Antibody Diluent 5B 1 x 6ml
    HIF1a Human Lyophilized Protein 2 x 25ng
    Plate Seals 1 unit
    Sample Diluent NS 1 x 12ml
    SimpleStep Pre-Coated 96-Well Microplate (ab206978) 1 unit
    Stop Solution 1 x 12ml
    TMB Substrate 1 x 12ml
  • 研究领域
  • 功能
    Functions as a master transcriptional regulator of the adaptive response to hypoxia. Under hypoxic conditions activates the transcription of over 40 genes, including, erythropoietin, glucose transporters, glycolytic enzymes, vascular endothelial growth factor, and other genes whose protein products increase oxygen delivery or facilitate metabolic adaptation to hypoxia. Plays an essential role in embryonic vascularization, tumor angiogenesis and pathophysiology of ischemic disease. Binds to core DNA sequence 5'-[AG]CGTG-3' within the hypoxia response element (HRE) of target gene promoters. Activation requires recruitment of transcriptional coactivators such as CREBPB and EP300. Activity is enhanced by interaction with both, NCOA1 or NCOA2. Interaction with redox regulatory protein APEX seems to activate CTAD and potentiates activation by NCOA1 and CREBBP.
  • 组织特异性
    Expressed in most tissues with highest levels in kidney and heart. Overexpressed in the majority of common human cancers and their metastases, due to the presence of intratumoral hypoxia and as a result of mutations in genes encoding oncoproteins and tumor suppressors.
  • 序列相似性
    Contains 1 basic helix-loop-helix (bHLH) domain.
    Contains 1 PAC (PAS-associated C-terminal) domain.
    Contains 2 PAS (PER-ARNT-SIM) domains.
  • 结构域
    Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID).
  • 翻译后修饰
    In normoxia, is hydroxylated on Pro-402 and Pro-564 in the oxygen-dependent degradation domain (ODD) by EGLN1/PHD1 and EGLN2/PHD2. EGLN3/PHD3 has also been shown to hydroxylate Pro-564. The hydroxylated prolines promote interaction with VHL, initiating rapid ubiquitination and subsequent proteasomal degradation. Deubiquitinated by USP20. Under hypoxia, proline hydroxylation is impaired and ubiquitination is attenuated, resulting in stabilization.
    In normoxia, is hydroxylated on Asn-803 by HIF1AN, thus abrogating interaction with CREBBP and EP300 and preventing transcriptional activation. This hydroxylation is inhibited by the Cu/Zn-chelator, Clioquinol.
    S-nitrosylation of Cys-800 may be responsible for increased recruitment of p300 coactivator necessary for transcriptional activity of HIF-1 complex.
    Requires phosphorylation for DNA-binding.
    Sumoylated; by SUMO1 under hypoxia. Sumoylation is enhanced through interaction with RWDD3. Desumoylation by SENP1 leads to increased HIF1A stability and transriptional activity.
    Ubiquitinated; in normoxia, following hydroxylation and interaction with VHL. Lys-532 appears to be the principal site of ubiquitination. Clioquinol, the Cu/Zn-chelator, inhibits ubiquitination through preventing hydroxylation at Asn-803.
    The iron and 2-oxoglutarate dependent 3-hydroxylation of asparagine is (S) stereospecific within HIF CTAD domains.
  • 细胞定位
    Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia.
  • Information by UniProt
  • 别名
    • ARNT interacting protein
    • ARNT-interacting protein
    • Basic helix loop helix PAS protein MOP1
    • Basic-helix-loop-helix-PAS protein MOP1
    • bHLHe78
    • Class E basic helix-loop-helix protein 78
    • HIF 1A
    • HIF 1alpha
    • HIF-1-alpha
    • HIF1
    • HIF1 A
    • HIF1 Alpha
    • HIF1-alpha
    • HIF1A
    • HIF1A_HUMAN
    • Hypoxia inducible factor 1 alpha
    • Hypoxia inducible factor 1 alpha isoform I.3
    • Hypoxia inducible factor 1 alpha subunit
    • Hypoxia inducible factor 1 alpha subunit basic helix loop helix transcription factor
    • Hypoxia inducible factor 1, alpha subunit (basic helix loop helix transcription factor)
    • Hypoxia inducible factor1alpha
    • Hypoxia-inducible factor 1-alpha
    • Member of PAS protein 1
    • Member of PAS superfamily 1
    • Member of the PAS Superfamily 1
    • MOP 1
    • MOP1
    • PAS domain-containing protein 8
    • PASD 8
    • PASD8
    see all
  • 数据库链接

相关产品

应用

Our Abpromise guarantee covers the use of ab171577 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

应用 Ab评论 说明
Sandwich ELISA Use at an assay dependent concentration.

图片

实验方案

文献

This product has been referenced in:
  • Reen FJ  et al. Bile signalling promotes chronic respiratory infections and antibiotic tolerance. Sci Rep 6:29768 (2016). Sandwich ELISA ; Human . Read more (PubMed: 27432520) »
  • Phelan JP  et al. Bile acids destabilise HIF-1a and promote anti-tumour phenotypes in cancer cell models. BMC Cancer 16:476 (2016). Sandwich ELISA ; Human . Read more (PubMed: 27416726) »

See all 2 Publications for this product

客户评价及客户问答

ab171577 - Human HIF-1-alpha ELISA Kit

Average Excellent 5/5 (Ease of Use)
Abreviews
I performed the determination of HiF 1 alpha on protein extracts from fresh tumor tissues and from tumor tissues frozen in order to verify if the method can be used in both situations.
I followed the procedure as in the protocol, without making any changes.
The standard curve was not reproducible with that of the datasheet and much lower values I obtained.
The values obtained from the samples tested are too low to make any assumption.
I attach scheme and values analysis.
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Miss. Rossella Galati

Verified customer

提交于 Mar 10 2016

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