Product datasheet
Anti-HIF-1-alpha antibody [H1alpha67] - ChIP Grade
ab1
29 Abreviews 46 References 10 Images
Overview
Product name
Anti-HIF-1-alpha antibody [H1alpha67] - ChIP Grade
Description
Mouse monoclonal [H1alpha67] to HIF-1-alpha - ChIP Grade
Specificity
HIF proteins play a central role in oxygen homeostasis in cells and are subject to rapid half-lives
and O2-dependent proteasomal degradation. HIF1 alpha may be degraded within 5-8 minutes
in both nuclear and cytoplasmic compartments (PMID: 11454738). We recommend using
treated cells as a positive control. For optimal results in WB Abcam recommends blocking for 1
hr in 5% milk and reducing the milk to 2% for the primary and secondary steps. We also
recommend TBST containing 0.05% Tween and reduced washing times (this antibody appears
to be sensitive to overwashing). In addition, a 2 hr primary incubation at RT rather than overnight
may improve results. For further information please contact our Scientific Support Team.
Tested applications
IHC-P, IP, ChIP, IHC-Fr, Flow Cyt, ICC/IF, WB
Species reactivity
Reacts with: Human
Predicted to work with: Mouse, Rat, Sheep, Rabbit, Cow, Pig, Ferret, Monkey
Immunogen
Fusion protein corresponding to Human HIF-1-alpha aa 400-550.
Positive control
This antibody gave a positive signal in whole cell (ab116322) and nuclear extracts of DFOtreated HeLa cells (ab180880). IHC-P on glioblastoma multiforme and also WB on nuclear
extracts of a hypoxically or CoCl2 induced cell line such as MCF-7,PC12, 293, or MEF.
General notes
For Mouse specific Hif-1-alpha rabbit monoclonal antibody, please see Ab179483 (clone ID:
EPR16897).
Ab179483 has been confirmed for Mouse sample in WB.
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
Constituent: PBS
Purity
Protein A purified
Clonality
Monoclonal
Clone number
H1alpha67
1
Isotype
IgG2b
Applications
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
IHC-P
Abreviews
Notes
Use at an assay dependent concentration. Perform heat mediated antigen
retrieval via the microwave method before commencing with IHC staining
protocol.
IP
Use a concentration of 5 µg/ml.
ChIP
Use at an assay dependent concentration.
IHC-Fr
Use at an assay dependent concentration.
Flow Cyt
Use at an assay dependent concentration. ab170192-Mouse monoclonal IgG2b,
is suitable for use as an isotype control with this antibody.
IF
1/100 - 1/200. PubMed: 25422886
ICC/IF
Use at an assay dependent concentration.
WB
Use a concentration of 5 µg/ml. Detects a band of approximately 120 kDa
(predicted molecular weight: 92 kDa).
For optimal results Abcam recommends blocking for 1 hr in 5% milk and
reducing the milk to 2% for the primary and secondary steps. We also
recommend TBST containing 0.05% Tween and reduced washing times (this
antibody appears to be sensitive to overwashing). We also recommend 2hr
primary incubation at RT rather than overnight. For further information please
contact our Scientific Support Team.
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.
2
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
modifications
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/Znchelator, 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.
Anti-HIF-1-alpha antibody [H1alpha67] - ChIP Grade images
3
All lanes : Anti-HIF-1-alpha antibody
[H1alpha67] - ChIP Grade (ab1) at 5 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma
cell line) Nuclear Lysate (ab150036)
Lane 2 : Hela-DFO treated (0.5mM, 24h)
Nuclear Lysate (ab180880)
Lysates/proteins at 40 µg per lane.
Secondary
Western blot - Anti-HIF-1-alpha [H1alpha67]
Goat Anti-Mouse IgG H&L (HRP)
antibody (ab1)
preadsorbed (ab97040) at 1/10000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 92 kDa
Observed band size : 110 kDa
Exposure time : 20 minutes
Abcam recommends using 5% milk as the
blocking agent, decreasing to 2% milk during
primary and secondary incubation. Abcam
welcomes customer feedback and would
appreciate any comments regarding this
product and the data presented above.
Secondary antibody - goat anti-mouse HRP
pre-adsorbed (ab97040)
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ab1 staining HIF-1-alpha in MCF7 cells
treated with metformin hydrochloride
(ab120847), by ICC/IF. Decrease in HIF-1alpha expression correlates with increased
concentration of metformin hydrochloride, as
described in literature.
Immunocytochemistry/ Immunofluorescence -
The cells were incubated at 37°C for 24h in
Anti-HIF-1-alpha [H1alpha67] antibody (ab1)
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 2h 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. A goat anti-mouse DyLight 488
antibody (ab96879) at 1/250 dilution was
used as the secondary antibody. Nuclei were
counterstained with DAPI and are shown in
blue.
ab1 staining HIF 1 alpha in Mouse skin tissue
sections by Immunohistochemistry (IHC-P paraformaldehyde-fixed, paraffin-embedded
sections). Tissue was fixed with
paraformaldehyde and blocked with 20%
serum for 30 minutes at 22°C; antigen
retrieval was by heat mediation in a citrate
buffer. Samples were incubated with primary
antibody (1/100) for 1 hour at 52°C. A HRPImmunohistochemistry (Formalin/PFA-fixed
conjugated Goat anti-mouse was used as the
paraffin-embedded sections) - Anti-HIF-1-alpha
secondary antibody.
antibody [H1alpha67] - ChIP Grade (ab1)
This image is courtesy of an Abreview submitted by
Miss. Virginia Gutiérrez
5
HIF-1-alpha was immunoprecipitated using
0.5mg HeLa Nuclear DFO treated whole cell
extract (ab180880), 5µg of Mouse
monoclonal to HIF-1-alpha and 50µl of protein
G magnetic beads (+). No antibody was
added to the control (-).
The antibody was incubated under agitation
with Protein G beads for 10min, HeLa DFO
treated whole cell extract lysate diluted in
Immunoprecipitation - Anti-HIF-1-alpha
[H1alpha67] antibody (ab1)
RIPA buffer was added to each sample and
incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS
loading buffer and incubated for 10min at
70°C; 10µl 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: 110kDa; HIF1 alpha
Flow cytometry using ab1. HeLa cells were
cultured untreated or with 1mM Deferoxamine
(ab120727) for 24 hours to induce HIF-1alpha protein levels. Cells were then
trypsinized, fixed with paraformaldehyde and
stained with ab1 (0.5 micrograms/mL). 1%
BSA in PBS was used as the blocking buffer
throughout. ab1 was labeled with and antiFlow Cytometry - Anti-HIF1 alpha antibody
mouse Alexa-488 dye. Unstained (black),
[H1alpha67] - ChIP Grade (ab1)
untreated (red) and DFO treated (blue) cell
traces are shown.
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ab1 staining HIF-1-alpha in mouse liver tissue
section by Immunohistochemistry (Frozen
sections). Tissue samples were fixed with
formaldehyde and permeablized with 0.2%
Triton-X100 before blocking with 2% BSA for
30 minutes at 20°C. The sample was
incubated with primary antibody (1/200) for 9
hours at 4°C. An Alexa Fluor®555-conjugated
Goat polyclonal to mouse IgG was used as
Immunohistochemistry (Frozen sections) - Anti-
secondary antibody at 1/200 dilution. DAPI
HIF-1-alpha [H1alpha67] antibody (ab1)
was used to stain the cell nuclei (blue).
This image is courtesy of an anonymous Abreview
All lanes : Anti-HIF-1-alpha antibody
[H1alpha67] - ChIP Grade (ab1) at 5 µg/ml
Lane 1 : Hela-Vehicle treated (Negative
Control) Whole Cell Lysate (ab116321)
Lane 2 : Hela-DFO treated (0.5mM, 24h)
Whole Cell Lysate (ab116322)
Lysates/proteins at 25 µg per lane.
Secondary
Western blot - Anti-HIF-1-alpha [H1alpha67]
Goat Anti-Mouse IgG H&L (HRP)
antibody (ab1)
preadsorbed (ab97040) at 1/5000 dilution
developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 92 kDa
Observed band size : 120 kDa
Additional bands at : 60 kDa. We are
unsure as to the identity of these extra bands.
Exposure time : 20 minutes
Secondary antibody - Goat anti-mouse HRP
predsorbed (ab97040)
7
ab1 staining HIF1 alpha in rat Infarct core
striate tissue sections by
Immunohistochemistry (IHC-P paraformaldehyde-fixed, paraffin-embedded
sections). Tissue was fixed with formaldehyde
and antigen retrieval was by heat mediation in
a citrate buffer. Samples were incubated with
primary antibody (1/1000) for 1 hour at 25°C.
A HRP-conjugated rabbit anti-mouse IgG
Immunohistochemistry (Formalin/PFA-fixed
whole molecule (1/200) was used as the
paraffin-embedded sections) - Anti-HIF-1-alpha
secondary antibody.
antibody [H1alpha67] - ChIP Grade (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
Immunocytochemistry/ Immunofluorescence -
hour at 25°C. The primary antibody was used
Anti-HIF-1-alpha [H1alpha67] antibody (ab1)
at 1/200 dilution in PBS and incubated with
This Image is courtesy of an anonymous Abreview
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.
Anti-HIF-1-alpha antibody [H1alpha67] - ChIP
Grade (ab1) at 1/400 dilution + Human Cell
lysate - whole cell (human lung
adenocarcinoma cell line ADLC-5M2) treated
for 16 hours with 100 micromolar
deferoxamine (DFO) at 20 µg
Performed under reducing conditions.
Western blot - Anti-HIF-1-alpha [H1alpha67]
antibody (ab1)
This image is taken from an Abreview submitted by
Mike Campa, no further information is known about
this image
Predicted band size : 92 kDa
Observed band size : 120 kDa
This image is taken from an Abreview
submitted by Mike Campa, no further
information is known about this image
PVDF membrane was used and blocked for
16 hours in 5% milk.
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"
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