Revision 14
#36169
Store at -20C
Orders:
877-616-CELL (2355)
Support:
877-678-TECH (8324)
3 Trask Lane | Danvers | Massachusetts | 01923 | USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:
W, W-F, W-S, IP, IF-IC, FC-FP, ChIP, ChIP-seq, C&R, C&T
Reactivity:
H M Mk
Sensitivity:
Endogenous
MW (kDa):
120
Source/Isotype:
Rabbit IgG
UniProt ID:
#Q16665
Entrez-Gene Id:
3091
Product Usage Information
The CUT&RUN dilution was determined using CUT&RUN Assay Kit #86652.
The CUT&Tag dilution was determined using CUT&Tag Assay Kit #77552.
| Application | Dilution |
|---|---|
| Western Blotting | 1:1000 |
| Fluorescent Western | 1:1000 |
| Simple Western™ | 1:10 - 1:50 |
| Immunoprecipitation | 1:50 |
| Immunofluorescence (Immunocytochemistry) | 1:400 - 1:1600 |
| Flow Cytometry (Fixed/Permeabilized) | 1:100 - 1:400 |
| Chromatin IP | 1:100 |
| Chromatin IP-seq | 1:100 |
| CUT&RUN | 1:100 |
| CUT&Tag | 1:100 |
Storage
Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/ml BSA, 50% glycerol and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.
For a carrier free (BSA and azide free) version of this product see product #36199.
For a carrier free (BSA and azide free) version of this product see product #36199.
Specificity/Sensitivity
HIF-1 alpha (D1S7W) Rabbit Monoclonal Antibody recognizes endogenous levels of total HIF-1α protein. This antibody does not cross-react with HIF-2α protein. Reactivity by immunofluorescence is human only.
Source / Purification
Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Leu478 of human HIF-1α protein.
Background
Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor that plays a critical role in the cellular response to hypoxia (1). The HIF1 complex consists of two subunits, HIF-1α and HIF-1β, which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family (2). HIF1 regulates the transcription of a broad range of genes that facilitate responses to the hypoxic environment, including genes regulating angiogenesis, erythropoiesis, cell cycle, metabolism, and apoptosis. The widely expressed HIF-1α is typically degraded rapidly in normoxic cells by the ubiquitin/proteasomal pathway. Under normoxic conditions, HIF-1α is proline hydroxylated leading to a conformational change that promotes binding to the von Hippel-Lindau protein (VHL) E3 ligase complex; ubiquitination and proteasomal degradation follows (3,4). Both hypoxic conditions and chemical hydroxylase inhibitors (such as desferrioxamine and cobalt) inhibit HIF-1α degradation and lead to its stabilization. In addition, HIF-1α can be induced in an oxygen-independent manner by various cytokines through the PI3K-AKT-mTOR pathway (5-7).
HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotic metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).
HIF-1β is also known as AhR nuclear translocator (ARNT) due to its ability to partner with the aryl hydrocarbon receptor (AhR) to form a heterodimeric transcription factor complex (8). Together with AhR, HIF-1β plays an important role in xenobiotic metabolism (8). In addition, a chromosomal translocation leading to a TEL-ARNT fusion protein is associated with acute myeloblastic leukemia (9). Studies also found that ARNT/HIF-1β expression levels decrease significantly in pancreatic islets from patients with type 2 diabetes, suggesting that HIF-1β plays an important role in pancreatic β-cell function (10).
Background References
- Sharp, F.R. and Bernaudin, M. (2004) Nat Rev Neurosci 5, 437-48.
- Wang, G.L. et al. (1995) Proc Natl Acad Sci U S A 92, 5510-4.
- Jaakkola, P. et al. (2001) Science 292, 468-72.
- Maxwell, P.H. et al. (1999) Nature 399, 271-5.
- Fukuda, R. et al. (2002) J Biol Chem 277, 38205-11.
- Jiang, B.H. et al. (2001) Cell Growth Differ 12, 363-9.
- Laughner, E. et al. (2001) Mol Cell Biol 21, 3995-4004.
- Walisser, J.A. et al. (2004) Proc Natl Acad Sci U S A 101, 16677-82.
- Salomon-Nguyen, F. et al. (2000) Proc Natl Acad Sci U S A 97, 6757-62.
- Gunton, J.E. et al. (2005) Cell 122, 337-49.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Western Blot Buffer
IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.
Applications Key
W: Western Blotting W-F: Fluorescent Western W-S: Simple Western™ IP: Immunoprecipitation IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized) ChIP: Chromatin IP ChIP-seq: Chromatin IP-seq C&R: CUT&RUN C&T: CUT&Tag
Cross-Reactivity Key
H: Human M: Mouse Mk: Monkey
Trademarks and Patents
Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.
使用に関する制限
使用に関する制限
法的な権限を与えられたCSTの担当者が署名した書面によって別途明示的に合意された場合を除き、CST、その関連会社または代理店が提供する製品には以下の条件が適用されます。お客様が定める条件でここに定められた条件に含まれるものを超えるもの、または、ここに定められた条件と異なるものは、法的な権限を与えられたCSTの担当者が別途書面にて受諾した場合を除き、拒絶され、いかなる効力も効果も有しません。
研究専用 (For Research Use Only) またはこれに類似する表示がされた製品は、 いかなる目的についても FDA または外国もしくは国内のその他の規制機関により承認、認可または許可を受けていません。 お客様は製品を診断もしくは治療目的で使用してはならず、また、製品に表示された内容に違反する方法で使用してはなりません。 CST が販売または使用許諾する製品は、エンドユーザーであるお客様に対し、使途を研究および開発のみに限定して提供されるものです。 診断、予防もしくは治療目的で製品を使用することまたは製品を再販売 (単独であるか他の製品等の一部であるかを問いません) もしくはその他の商業的利用の目的で購入することについては、CST から別途許諾を得る必要があります。 お客様は以下の事項を遵守しなければなりません。(a) CST の製品 (単独であるか他の資材と一緒であるかを問いません) を販売、使用許諾、貸与、寄付もしくはその他の態様で第三者に譲渡したり使用させたりしてはなりません。また、商用の製品を製造するために CST の製品を使用してはなりません。(b) 複製、改変、リバースエンジニアリング、逆コンパイル、 分解または他の方法により製品の構造または技術を解明しようとしてはなりません。また、 CST の製品またはサービスと競合する製品またはサービスを開発する目的で CST の製品を使用してはなりません。(c) CST の製品の商標、商号、ロゴ、特許または著作権に関する通知または表示を除去したり改変したりしてはなりません。(d) CST の製品をCST 製品販売条件(CST’s Product Terms of Sale) および該当する書面のみに従って使用しなければなりません。(e) CST の製品に関連してお客様が使用する第三者の製品またはサービスに関する使用許諾条件、 サービス提供条件またはこれに類する合意事項を遵守しなければなりません。
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 14
Western blot analysis of extracts from Hep G2 cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +), Raji cells untreated (-) or treated with cobalt chloride (100 µM, 4 h; +) and U-2 OS cells untreated (-) or treated with DMOG (1 mM, 6 h; +) using HIF-1α (D1S7W) XP® Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western blot analysis of extracts from Hep G2 cells, untreated (-) or treated with cobalt chloride (100 μM, 24 hr; +), using HIF-1α (D1S7W) XP® Rabbit mAb #36169 (green), and β-Actin (8H10D10) Mouse mAb #3700 (red). Anti-rabbit IgG (H+L) (DyLight 800 4X PEG Conjugate) #5151 (green) and Anti-mouse IgG (H+L) (DyLight 680 Conjugate) #5470 (red) were used as secondary antibodies.
Simple Western™ analysis of lysates (0.1 mg/mL) from HepG2 cells treated with cobalt chloride (100 µM, 24 hr) using HIF-1α (D1S7W) XP® Rabbit mAb #36169. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 14
Immunoprecipitation of HIF-1α from lysate of Hep G2 cells treated with cobalt chloride (100 µM, 4 h). Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is HIF-1α (D1S7W) XP® Rabbit mAb. Western blot analysis was performed using HIF-1α (D1S7W) XP® Rabbit mAb. Anti-rabbit IgG, HRP-linked Antibody #7074 was used as the secondary antibody.
Confocal immunofluorescent analysis of Hep G2 cells, untreated (left) or treated with cobalt chloride (500 μM, 24 h; right), using HIF-1α (D1S7W) XP® Rabbit mAb (green). Actin filaments were labeled with DyLight™ 554 Phalloidin #13054 (red).
Flow cytometric analysis of U-2 OS cells, untreated (blue) or treated with DMOG (1 mM, 6 h; green), using HIF-1α (D1S7W) XP® Rabbit mAb (solid lines) or concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype control #3900 (dashed lines). Anti-rabbit IgG (H+L), F(ab')2 Fragment (Alexa Fluor® 488 Conjugate) #4412 was used as a secondary antibody.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 14
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across ARRDC3, a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across chromosome 5 (upper), including ARRDC3 (lower), a known target gene of HIF-1α (see additional figure containing ChIP-qPCR data).
Chromatin immunoprecipitations were performed with cross-linked chromatin from MCF7 cells treated with cobalt chloride (100 μM, overnight) and either HIF-1α (D1S7W) XP® Rabbit mAb or Normal Rabbit IgG #2729, using SimpleChIP® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. The enriched DNA was quantified by real-time PCR using human ARRDC3 downstream primers, SimpleChIP® Human ERRFI1 Upstream Primers #31180, and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 14
CUT&RUN was performed with MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA library was prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figure shows binding across STC2, a known target gene of HIF-1α (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb, using CUT&RUN Assay Kit #86652. DNA Libraries were prepared using DNA Library Prep Kit for Illumina® (ChIP-seq, CUT&RUN) #56795. The figures show binding across chromosome 5 (upper), including STC2 (lower), a known target gene of HIF-1α (see additional figure containing CUT&RUN-qPCR data).
CUT&RUN was performed with MCF7 cells treated with cobalt chloride (100 μM) overnight and HIF-1α (D1S7W) XP® Rabbit mAb or Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, using CUT&RUN Assay Kit #86652. The enriched DNA was quantified by real-time PCR using human STC2 exon 1 primers, human FAM162A promoter primers and SimpleChIP® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin, which is equivalent to one.
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.
Revision 14
CUT&Tag was performed with MCF7 cells treated with cobalt chloride (100uM) overnight and HIF-1 alpha (D1S7W) Rabbit Monoclonal Antibody, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across the BNIP3 gene.
CUT&Tag was performed with MCF7 cells treated with cobalt chloride (100uM) overnight and HIF-1 alpha (D1S7W) Rabbit Monoclonal Antibody, using CUT&Tag Assay Kit #77552. DNA library was prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figures show binding across chromosome 10 (upper), including the BNIP3 gene (lower).
Orders: 877-616-CELL (2355) • [email protected] • Support: 877-678-TECH (8324) • [email protected] •
Web:
cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.