Hypoxia Inducible Factor 1, alpha Subunit (Basic Helix-Loop-Helix Transcription Factor) (HIF1A) (AA 432-528) antibody

Details for Product No. ABIN151033
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Antigen
Synonyms hif1a, AA959795, HIF1alpha, MOP1, bHLHe78, HIF-1A, HIF-1alpha, HIF1, HIF1-ALPHA, PASD8, HIF1-alpha, hif-1a
Epitope
AA 432-528
(41), (39), (23), (11), (9), (8), (8), (7), (7), (6), (6), (6), (6), (5), (2), (2), (2), (2), (2), (2), (2), (2), (2), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1)
Reactivity
Cow (Bovine), Ferret, Human, Monkey, Mouse (Murine), Pig (Porcine), Rabbit, Rat (Rattus), Sheep (Ovine)
(260), (112), (76), (54), (36), (31), (21), (16), (16), (15), (10), (3), (2), (1), (1), (1)
Host
Mouse
(188), (126), (2)
Clonality (Clone)
Monoclonal ()
Conjugate
Un-conjugated
(27), (15), (12), (12), (12), (6), (3), (3), (2), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1), (1)
Application
Western Blotting (WB), Chromatin Immunoprecipitation (ChIP), ELISA, Flow Cytometry (FACS), Gel Shift (GS), Immunocytochemistry (ICC), Immunofluorescence (IF), Immunohistochemistry (IHC), Immunohistochemistry (Frozen Sections) (IHC (fro)), Immunohistochemistry (Paraffin-embedded Sections) (IHC (p)), Immunoprecipitation (IP)
(235), (98), (91), (82), (78), (68), (46), (39), (13), (12), (10), (10), (9), (4), (3), (1), (1)
Pubmed 28 references available
Quantity 0.025 mL
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Catalog No. ABIN151033
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Immunogen Fusion protein containing amino acids 432-528 of human HIF-1 alpha.
Clone H1alpha67
Isotype IgG2b
Cross-Reactivity (Details) Reactivity with Candida albicans reported by a customer review. Reactivity with Xenopus reported in the scientific literature (PMID: 18303027).
Purification Protein G purified
Alternative Name HIF1A / HIF1 alpha
Background Hypoxia contributes significantly to the pathophysiology of major categories of humandisease, including myocardial and cerebral ischemia, cancer, pulmonary hypertension,congenital heart disease and chronic obstructive pulmonary disease.HIF-1 is a nuclear protein involved in mammalian oxygen homeostasis. This occurs asa post-translational modification by prolyl hydroxylation. HIF-1 is a heterodimercomposed of HIF-1 alpha and HIF-1 beta subunits. Both subunits are constantlytranslated. However, under normoxic conditions, human HIF-1 alpha is hydroxylated atPro402 or Pro564 by a set of HIF prolyl hydroxylases, is polyubiquinated, and eventuallydegraded in proteosomes. Under hypoxic conditions, the lack of hydroxylation preventsHIF degradation and increases transcriptional activity. Therefore, the concentration ofHIF-1 alpha increases in the cell. In contrast, HIF-1 beta remains stable under eithercondition. HIF hydroxylases provide insight into hypoxic cell responses, which may beused to help isolate therapeutic targets. Alternate Names: anti-Hypoxia-inducible factor 1 alpha antibody, anti-HIF1 alpha antibody, anti-ARNTinteracting protein antibody, anti-Hif1a antibody, anti-ARNT interacting protein antibody,anti-HIF-1alpha antibody, anti-Hypoxia inducible factor 1 alpha antibody, anti-Hypoxiainducible factor 1 alpha subunit basic helix antibody.
Gene Symbol: HIF1A
Gene ID 3091
UniProt Q16665
Application Notes This HIF-1 alpha (H1alpha67) antibody is useful for Chromatin Immunoprecipitation (PMID: 21871655), ELISA (PMID: 20042684), Flow Cytometry, Gel Super Shift Assays, Immunocytochemistry/Immunofluorescence, Immunohistochemistry on frozen and paraffin-embedded sections, Immunoprecipitation and Western Blot. In WB, a band can be seen at 120 kDa representing HIF-1 alpha in induced tissues and cells. Multiple bands may be seen at 100-120 kDa representing post-translational modification of HIF-1 alpha. For WB, testing on nuclear extracts is recommended. This antibody has been used to immunoprecipitate human HIF-1 alpha. For ChIP, refer to research papers with PubMed ID 16204079 and 21871655. This product has been cited for Gel Super Shift Assays in PubMed ID 22411794.
Recommended dilutions: Chromatin Immunoprecipitation 1:10-1:500, ELISA, Flow Cytometry, Gel Super Shift Assays 1:1-1:100, Immunocytochemistry/Immunofluorescence 1:50, Immunohistochemistry 1:20-1:50, Immunohistochemistry-Frozen 1:20-1:50, Immunohistochemistry-Paraffin 1:20-1:50, Immunoprecipitation 1:10-1:500, Western Blot 1:500
Protocol Western Blot Protocols specific for HIF-1 alpha Antibody HIF-1 alpha Western Blot General Information:
1. The HIF proteins are among the most rapidly degrading proteins ever studied. Upon cellular re-oxygenation it can be completely degraded in less than 1 minute. Therefore, it is critical to prep only a few plates/dishes/flasks of cells at a time and to immediately place the cells into ice cold buffers and perform the whole protein prep on ice.
. HIF-1 is largely undetectable in cells or tissues grown under normoxic conditions. It is stabilized only at O2 concentrations below 5 % or with treatment using certain agents (CoCl2, DFO, etc.) so proper sample preparation is critical.
. Upon stabilization HIF-1 translocates to the nucleus. The best western blots (cleanest) are always done using nuclear extracts. It is possible to detect HIF-1 in whole cell extracts, but they tend to be much dirtier and the staining is much weaker.
. Finally, we recommend that a positive/negative control always be run side by side so that it is possible to discern which band is upregulated in the hypoxic sample. Unprocessed HIF1 is ~95 kDa while the fully post-translationally modified form is ~116 kDa, or larger. Additionally, HIF-1 alpha may form a heterodimer with HIF-1 beta (Duan, et al. Circulation. 2005,111:2227-2232.).Depending on the sample, treatment, etc. you may see either a band or a doublet."EPO transcription can be activated by exposure of Hep3B cells to either hypoxia or cobalt chloride (7). HIF-1 binding activity was induced after 1 h and was maximal after 4-h treatment of Hep3B cells with 75 ,M cobalt chloride (Fig. 2A), which is similar to the kinetics of HIF-1 induction by hypoxia (data not shown). Exposure of HeLa cells to cobalt chloride for 4 h also induced HIF-1 activity. In contrast to hypoxia, which induced a doublet band corresponding to HIF-1 in EMSAs, cobalt chloride induced a single band of HIF-1 activity in both Hep3B and HeLa cells (compare Figs. 1A and 2A). We have not determined the basis for this reproducible difference in response to stimulation by hypoxia as compared to cobalt chloride" (Wang G, et al. (1993) PNAS 90, 4304-4308.).Thus, it is critical to be able to look at upregulation compared to the control.Western Blot Protocol 1 (used to produce the image on the datasheet)
. Perform SDS-PAGE (3-8 %) on samples to be analyzed, loading 40ug of total protein per lane (COS-7 treated and untreated lysates.
. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.
. Stain the blot using ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
. Rinse the blot in TBS for approximately 5 minutes.
. Block the membrane using 5 % non-fat dry milk in TBS for 1 hour.
. Dilute the mouse anti-HIF-1 alpha primary antibody in blocking buffer and incubate 2 hours at room temperature.
. Wash the membrane in water for 5 minutes and apply the diluted mouse-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) and incubate 1 hour at room temperature.
. Wash the blot in TBS containing 0.05-0.1 % Tween-20 for 10-20 minutes.
. Wash the blot in type I water for an additional 10-20 minutes (this step can be repeated as required to reduce background.
. Apply the detection reagent of choice in accordance with the manufacturers instructions (Amersham ECL is the standard reagent used).Note: Tween-20 can be added to the blocking buffer at a final concentration of 0.05-0.2 %, provided it does not interfere with antibody-antigen binding. Western Blot Procedure 2
Restrictions For Research Use only
Format Liquid
Concentration 1.2 mg/mL
Buffer PBS, Sodium Azide
Preservative Sodium azide
Precaution of Use WARNING: Reagents contain sodium azide. Sodium azide is very toxic if ingested or inhaled. Avoid contact with skin, eyes, or clothing. Wear eye or face protection when handling. If skin or eye contact occurs, wash with copious amounts of water. If ingested or inhaled, contact a physician immediately. Sodium azide yields toxic hydrazoic acid under acidic conditions. Dilute azide-containing compounds in running water before discarding to avoid accumulation of potentially explosive deposits in lead or copper plumbing.
Handling Advice Do not freeze.
Storage 4 °C
Supplier Images
anti-Hypoxia Inducible Factor 1, alpha Subunit (Basic Helix-Loop-Helix Transcription Factor) (HIF1A) (AA 432-528) antibody Western analysis of COS-7 nuclear extracts (50 ug) using ABIN151032.
General Gothiue, Richard, Berra et al.: "Identification of alternative spliced variants of human hypoxia-inducible factor-1alpha." in: The Journal of biological chemistry, Vol. 275, Issue 10, pp. 6922-7, 2000 (PubMed).

Birner, Schindl, Obermair et al.: "Overexpression of hypoxia-inducible factor 1alpha is a marker for an unfavorable prognosis in early-stage invasive cervical cancer." in: Cancer research, Vol. 60, Issue 17, pp. 4693-6, 2000 (PubMed).

Birner, Schindl, Obermair et al.: "Expression of hypoxia-inducible factor 1alpha in epithelial ovarian tumors: its impact on prognosis and on response to chemotherapy." in: Clinical cancer research : an official journal of the American Association for Cancer Research, Vol. 7, Issue 6, pp. 1661-8, 2001 (PubMed).

Birner, Gatterbauer, Oberhuber et al.: "Expression of hypoxia-inducible factor-1 alpha in oligodendrogliomas: its impact on prognosis and on neoangiogenesis." in: Cancer, Vol. 92, Issue 1, pp. 165-71, 2001 (PubMed).

Duyndam, Hulscher, Fontijn et al.: "Induction of vascular endothelial growth factor expression and hypoxia-inducible factor 1alpha protein by the oxidative stressor arsenite." in: The Journal of biological chemistry, Vol. 276, Issue 51, pp. 48066-76, 2001 (PubMed).

Aminova, Chavez, Lee et al.: "Prosurvival and prodeath effects of hypoxia-inducible factor-1alpha stabilization in a murine hippocampal cell line." in: The Journal of biological chemistry, Vol. 280, Issue 5, pp. 3996-4003, 2005 (PubMed).

Alberghini, Recalcati, Tacchini et al.: "Loss of the von Hippel Lindau tumor suppressor disrupts iron homeostasis in renal carcinoma cells." in: The Journal of biological chemistry, Vol. 280, Issue 34, pp. 30120-8, 2005 (PubMed).

Higami, Barger, Page et al.: "Energy restriction lowers the expression of genes linked to inflammation, the cytoskeleton, the extracellular matrix, and angiogenesis in mouse adipose tissue." in: The Journal of nutrition, Vol. 136, Issue 2, pp. 343-52, 2006 (PubMed).

Galabova-Kovacs, Matzen, Piazzolla et al.: "Essential role of B-Raf in ERK activation during extraembryonic development." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 103, Issue 5, pp. 1325-30, 2006 (PubMed).

Hsu, Adereth, Kose et al.: "Endocytic function of von Hippel-Lindau tumor suppressor protein regulates surface localization of fibroblast growth factor receptor 1 and cell motility." in: The Journal of biological chemistry, Vol. 281, Issue 17, pp. 12069-80, 2006 (PubMed).

Hui, Bauer, Striet et al.: "Calcium signaling stimulates translation of HIF-alpha during hypoxia." in: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 20, Issue 3, pp. 466-75, 2006 (PubMed).

Fath, Kong, Liang et al.: "Histone deacetylase inhibitors repress the transactivation potential of hypoxia-inducible factors independently of direct acetylation of HIF-alpha." in: The Journal of biological chemistry, Vol. 281, Issue 19, pp. 13612-9, 2006 (PubMed).

Franco, Man, Chen et al.: "Targeted anti-vascular endothelial growth factor receptor-2 therapy leads to short-term and long-term impairment of vascular function and increase in tumor hypoxia." in: Cancer research, Vol. 66, Issue 7, pp. 3639-48, 2006 (PubMed).

Hu, Iyer, Sataur et al.: "Differential regulation of the transcriptional activities of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha in stem cells." in: Molecular and cellular biology, Vol. 26, Issue 9, pp. 3514-26, 2006 (PubMed).

Bracken, Fedele, Linke et al.: "Cell-specific regulation of hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha stabilization and transactivation in a graded oxygen environment." in: The Journal of biological chemistry, Vol. 281, Issue 32, pp. 22575-85, 2006 (PubMed).

Bernhardt, Cuampean, Kany et al.: "Preconditional activation of hypoxia-inducible factors ameliorates ischemic acute renal failure." in: Journal of the American Society of Nephrology : JASN, Vol. 17, Issue 7, pp. 1970-8, 2006 (PubMed).

Chade, Zhu, Mushin et al.: "Simvastatin promotes angiogenesis and prevents microvascular remodeling in chronic renal ischemia." in: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 20, Issue 10, pp. 1706-8, 2006 (PubMed).

Asikainen, Chang, Coalson et al.: "Improved lung growth and function through hypoxia-inducible factor in primate chronic lung disease of prematurity." in: The FASEB journal : official publication of the Federation of American Societies for Experimental Biology, Vol. 20, Issue 10, pp. 1698-700, 2006 (PubMed).

Grimmer, Balbus, Lang et al.: "Regulation of type II collagen synthesis during osteoarthritis by prolyl-4-hydroxylases: possible influence of low oxygen levels." in: The American journal of pathology, Vol. 169, Issue 2, pp. 491-502, 2006 (PubMed).

Brooks, Wang, Yang et al.: "Characterization of cell clones isolated from hypoxia-selected renal proximal tubular cells." in: American journal of physiology. Renal physiology, Vol. 292, Issue 1, pp. F243-52, 2007 (PubMed).

Carroll, Kenerson, Yeung et al.: "Latent Kaposi's sarcoma-associated herpesvirus infection of endothelial cells activates hypoxia-induced factors." in: Journal of virology, Vol. 80, Issue 21, pp. 10802-12, 2006 (PubMed).

Chavez, Baranova, Lin et al.: "The transcriptional activator hypoxia inducible factor 2 (HIF-2/EPAS-1) regulates the oxygen-dependent expression of erythropoietin in cortical astrocytes." in: The Journal of neuroscience : the official journal of the Society for Neuroscience, Vol. 26, Issue 37, pp. 9471-81, 2006 (PubMed).

Chen, Yan, Davidson et al.: "Hypoxic stress induces dimethylated histone H3 lysine 9 through histone methyltransferase G9a in mammalian cells." in: Cancer research, Vol. 66, Issue 18, pp. 9009-16, 2006 (PubMed).

Groenman, Rutter, Caniggia et al.: "Hypoxia-inducible factors in the first trimester human lung." in: The journal of histochemistry and cytochemistry : official journal of the Histochemistry Society, Vol. 55, Issue 4, pp. 355-63, 2007 (PubMed).

Creighton-Gutteridge, Cardellina, Stephen et al.: "Cell type-specific, topoisomerase II-dependent inhibition of hypoxia-inducible factor-1alpha protein accumulation by NSC 644221." in: Clinical cancer research : an official journal of the American Association for Cancer Research, Vol. 13, Issue 3, pp. 1010-8, 2007 (PubMed).

Bernhardt, Wiesener, Weidemann et al.: "Involvement of hypoxia-inducible transcription factors in polycystic kidney disease." in: The American journal of pathology, Vol. 170, Issue 3, pp. 830-42, 2007 (PubMed).

Chin, Jiang, Wegiel et al.: "Hypoxia-inducible factor 1alpha stabilization by carbon monoxide results in cytoprotective preconditioning." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 104, Issue 12, pp. 5109-14, 2007 (PubMed).

Bateman, Tokunaga, Kareco et al.: "Myocardial hypoxia-inducible HIF-1alpha, VEGF, and GLUT1 gene expression is associated with microvascular and ICAM-1 heterogeneity during endotoxemia." in: American journal of physiology. Heart and circulatory physiology, Vol. 293, Issue 1, pp. H448-56, 2007 (PubMed).

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