Anti-HIF-1 alpha antibody [H1alpha67] (ab1)
Key features and details
- Mouse monoclonal [H1alpha67] to HIF-1 alpha
- Suitable for: IP, Flow Cyt, ICC/IF, WB
- Reacts with: Human
- Isotype: IgG2b
Related conjugates and formulations
Overview
-
Product name
Anti-HIF-1 alpha antibody [H1alpha67]
See all HIF-1 alpha primary antibodies -
Description
Mouse monoclonal [H1alpha67] to HIF-1 alpha -
Host species
Mouse -
Tested applications
Suitable for: IP, Flow Cyt, ICC/IF, WBmore details
Unsuitable for: IHC-Fr or IHC-P -
Species reactivity
Reacts with: Human
Predicted to work with: Mouse, Rat -
Immunogen
Recombinant fragment. This information is proprietary to Abcam and/or its suppliers.
-
Positive control
- WB: HeLa (DFO treated 0.5mM, 24h) nuclear lysate (ab180880); Human whole cell lysate (human lung adenocarcinoma cell line ADLC-5M2) (DFO treated 100uM, 16h). IP: HeLa (DFO treated 0.5 mg) nuclear lysate. ICC/F: MCF7 cells. Flow Cyt: HeLa (Human epithelial cell line from cervix adenocarcinoma) cells treated with 1mM Deferoxamine (ab120727) for 24 hours.
-
General notes
For WB, we recommend using positive control samples such as DFO or CoCl2 treated nulcear cell lysates such as ab180880. Ensure cell lysis occurs quickly (within 2 mins) if removed from hypoxia. Loading a high amount of sample (>50 µg) and addition of protease inhibitors (e.g. ab65621) may also enhance detection.
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com.
The Life Science industry has been in the grips of a reproducibility crisis for a number of years. Abcam is leading the way in addressing this with our range of recombinant monoclonal antibodies and knockout edited cell lines for gold-standard validation. Please check that this product meets your needs before purchasing.
If you have any questions, special requirements or concerns, please send us an inquiry and/or contact our Support team ahead of purchase. Recommended alternatives for this product can be found below, along with publications, customer reviews and Q&As
Properties
-
Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine -
Concentration information loading...
-
Purity
Protein G purified -
Clonality
Monoclonal -
Clone number
H1alpha67 -
Isotype
IgG2b -
Research areas
Associated products
-
Alternative Versions
-
Assay kits
-
ChIP Related Products
-
Compatible Secondaries
-
Isotype control
-
Positive Controls
-
Recombinant Protein
-
Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab1 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Application | Abreviews | Notes |
---|---|---|
IP | (2) |
Use a concentration of 5 µg/ml.
|
Flow Cyt |
Use at an assay dependent concentration.
ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody. |
|
ICC/IF | (6) |
1/100 - 1/200.
PubMed: 25422886 We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody. |
WB | (22) |
Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa).
We recommend blocking for 1 hour with 5% milk in TBST and reducing to 2% milk in TBST for the primary and secondary antibody incubation steps. For primary antibody incubation, we recommend 2 hours at room temperature. We recommend Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody. |
Notes |
---|
IP
Use a concentration of 5 µg/ml. |
Flow Cyt
Use at an assay dependent concentration. ab170192 - Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody. |
ICC/IF
1/100 - 1/200. PubMed: 25422886 We recommend Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) secondary antibody. |
WB
Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa (predicted molecular weight: 92 kDa). We recommend blocking for 1 hour with 5% milk in TBST and reducing to 2% milk in TBST for the primary and secondary antibody incubation steps. For primary antibody incubation, we recommend 2 hours at room temperature. We recommend Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody. |
Target
-
Function
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. -
Tissue specificity
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. -
Sequence similarities
Contains 1 basic helix-loop-helix (bHLH) domain.
Contains 1 PAC (PAS-associated C-terminal) domain.
Contains 2 PAS (PER-ARNT-SIM) domains. -
Domain
Contains two independent C-terminal transactivation domains, NTAD and CTAD, which function synergistically. Their transcriptional activity is repressed by an intervening inhibitory domain (ID). -
Post-translational
modificationsIn 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. -
Cellular localization
Cytoplasm. Nucleus. Cytoplasmic in normoxia, nuclear translocation in response to hypoxia. Colocalizes with SUMO1 in the nucleus, under hypoxia. - Information by UniProt
-
Database links
- Entrez Gene: 3091 Human
- Entrez Gene: 15251 Mouse
- Entrez Gene: 29560 Rat
- Omim: 603348 Human
- SwissProt: Q16665 Human
- SwissProt: Q61221 Mouse
- SwissProt: O35800 Rat
- Unigene: 597216 Human
see all -
Alternative names
- ARNT interacting protein antibody
- ARNT-interacting protein antibody
- Basic helix loop helix PAS protein MOP1 antibody
see all
Images
-
All lanes : Anti-HIF-1 alpha antibody [H1alpha67] (ab1) at 5 µg/ml
Lane 1 :HeLa nuclear extract lysate (ab150036) at 40 µg
Lane 2 :Hela-DFO treated (0.5mM, 24h) Nuclear Lysate (ab180880) at 40 µg
Lane 3 : HeLa nuclear control at 40 µg
Lane 4 : HeLa nuclear DFO treated at 40 µg
Lane 5 :Recombinant Human HIF-1 alpha protein (ab154478) at 0.001 µg
Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 92 kDa
Exposure time: 20 minutesWe recommend using 5% milk in TBST as the blocking agent, decreasing to 2% milk in TBST during primary and secondary antibody incubation.
Blots were developed with Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) secondary antibody
-
HIF-1 alpha was immunoprecipitated using 0.5 mg HeLa Nuclear DFO treated whole cell extract (ab180880), 5 µg of Mouse monoclonal to HIF-1 alpha and 50 µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10 minutes, HeLa DFO treated whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10 minutes under agitation.
Proteins were eluted by addition of 40 µl SDS loading buffer and incubated for 10 minutes at 70°C; 10 µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab1.
Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1:20,000 dilution.
Band: 110 kDa; HIF1 alpha
-
Western blot - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)This image is taken from an Abreview submitted by Mike Campa, no further information is known about this imageAnti-HIF-1 alpha antibody [H1alpha67] (ab1) at 1/400 dilution + Human whole cell lysate (human lung adenocarcinoma cell line ADLC-5M2) treated for 16 hours with 100 micromolar deferoxamine (DFO) at 20 µg
Performed under reducing conditions.
Predicted band size: 92 kDa
Observed band size: 120 kDa why is the actual band size different from the predicted?
PVDF membrane was used and blocked for 16 hours in 5% milk. -
ab1 staining HIF-1-alpha in MCF7 (Human breast adenocarcinoma cell line) cells treated with metformin hydrochloride ab12084, by ICC/IF. Decrease in HIF-1-alpha expression correlates with increased concentration of metformin hydrochloride, as described in literature.
The cells were incubated at 37°C for 24 hours in media containing different concentrations of ab120847 (metformin hydrochloride) in water, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2 hours at room temperature. Staining of the treated cells with ab1 (10 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed (ab96879) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue. -
Immunocytochemistry/ Immunofluorescence - Anti-HIF-1 alpha antibody [H1alpha67] (ab1)This Image is courtesy of an anonymous Abreview
ab1 at 1/200 dilution staining HIF-1 alpha in human 293FT cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde, permeabilized in 0.5% Trition X-100 and blocked in 5% BSA for 1 hour at 25°C. The primary antibody was used at 1/200 dilution in PBS and incubated with sample at 4°C for 12 hours. An Alexa Fluor® 488 conjugated Goat polyclonal to mouse IgG was used as secondary at 1/500 dilution.
-
Flow cytometry using ab1. HeLa (Human epithelial cell line from cervix adenocarcinoma) cells were cultured untreated or with 1mM Deferoxamine (ab120727) for 24 hours to induce HIF-1-alpha protein levels. Cells were then trypsinized, fixed with paraformaldehyde and stained with ab1 (0.5 µg/mL). 1% BSA in PBS was used as the blocking buffer throughout. ab1 was labeled with and anti-mouse Alexa-Fluor® 488 dye. Unstained (black), untreated (red) and DFO treated (blue) cell traces are shown.
Protocols
Datasheets and documents
-
SDS download
-
Datasheet download
References (340)
ab1 has been referenced in 340 publications.
- Wen M et al. HIF-1a mediates the protective effect of plasma extracellular particles induced by remote ischaemic preconditioning on oxidative stress injury in human umbilical vein endothelial cells. Exp Ther Med 23:48 (2022). PubMed: 34917179
- Zhang M et al. Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1β. BMC Pharmacol Toxicol 23:26 (2022). PubMed: 35473600
- Guo C et al. miR-199a-5p Relieves Obstructive Sleep Apnea Syndrome-Related Hypertension by Targeting HIF-1α. J Immunol Res 2022:7236647 (2022). PubMed: 35935584
- Wang X et al. miR‑519d‑3p released by human blastocysts negatively regulates endometrial epithelial cell adhesion by targeting HIF1α. Int J Mol Med 50:N/A (2022). PubMed: 35959792
- Kij A et al. Thrombin Inhibition Prevents Endothelial Dysfunction and Reverses 20-HETE Overproduction without Affecting Blood Pressure in Angiotensin II-Induced Hypertension in Mice. Int J Mol Sci 22:N/A (2021). PubMed: 34445374