HIF-1 alpha Antibody

Images

 
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-479] - Analysis in A. PC12 (rat) cells, 20% oxygen and B. PC12 (rat) cells, 1% oxygen.
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-479] - Analysis of HIF-1 alpha in mouse hepatocytes (left lane: normoxic and right lane: hypoxic) using HIF-1 alpha antibody. Image from verified ...read more
Biological Strategies: Western Blot: HIF-1 alpha Antibody [NB100-479] - Analysis in a hypoxic sample. Lane 1: CoCl2 treated Cos-7 nuclear extract (50 ug, hypoxic). Lane 2: Untreated Cos-7 nuclear extract (50 ...read more
Immunohistochemistry: HIF-1 alpha Antibody [NB100-479] - Staining of human placenta, villi.
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Staining in normal mouse kidney showing (A) staining with antibody and (B) blocking peptide used at 10-fold excess concentration. Images provided by Dr ...read more
Immunocytochemistry/ Immunofluorescence: HIF-1 alpha Antibody [NB100-479] - Detection of HIF-1 Alpha (green) in RCC4 cells using NB100-479. Nuclei (Blue) were counterstained using Hoechst 33258.
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Analysis in paraffin section of mouse skin wound using NB100-479 at 1:6,000. Image courtesy of product review by Ian Darby of RMIT University.
Western Blot: HIF-1 alpha Antibody [NB100-479] - Comparison of HIF-1 alpha levels in HMC-1.2 cells and CBMC cells under normoxic and hopoxic conditions. DFX was used as a positive control. [PMID: 20808808] Image using ...read more
Western Blot: HIF-1 alpha Antibody [NB100-479] - U251 cells exposed to 4 hour hypoxia (0.5% oxygen) incubation. This image was submitted via customer Review.
Immunohistochemistry: HIF-1 alpha Antibody [NB100-479] - Analysis in normal mouse kidney. Image courtesy of product review by Yves Heremans.
Immunohistochemistry-Paraffin: HIF-1 alpha Antibody [NB100-479] - Analysis in human kidney tumor.

Product Details

Summary
Reactivity Hu, Mu, Rt, Fi, Ha, Pm, RbSpecies Glossary
Applications WB, ChIP, Flow, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IP
Clonality
Polyclonal
Host
Rabbit
Conjugate
Unconjugated
Concentration
1.0 mg/ml
Validated by:
   

Biological Strategies

     

HIF-1 alpha Antibody Summary

Immunogen
Fusion protein including amino acids 530 - 825 of the mouse HIF-1 alpha protein [UniProt# Q61221].
Isotype
IgG
Clonality
Polyclonal
Host
Rabbit
Gene
HIF1A
Purity
Immunogen affinity purified
Innovator's Reward
Test in a species/application not listed above to receive a full credit towards a future purchase.

Applications/Dilutions

Dilutions
  • Western Blot
  • Chromatin Immunoprecipitation 1:10 - 1:500
  • Flow Cytometry 1:10 - 1:1000
  • Immunoblotting
  • Immunocytochemistry/Immunofluorescence 1:100
  • Immunohistochemistry 5-10 ug/ml
  • Immunohistochemistry-Frozen 5-10 ug/ml
  • Immunohistochemistry-Paraffin 5-10 ug/ml
  • Immunoprecipitation 1:10 - 1:500
Application Notes
WB recognizes a band in hypoxic samples at ~115 kDa. HIF-1 alpha protein (NBC1-18422) has been successfully used for antibody preadsorption/blocking assay in IHC-P with HIF-1 alpha antibody NB100-479. Flow Cytometry usage reported in scientific literature (PMID 21917971). Use in Immunoblotting reported in scientific literature (PMID 28506759).
Theoretical MW
93 kDa.
Disclaimer note: The observed molecular weight of the protein may vary from the listed predicted molecular weight due to post translational modifications, post translation cleavages, relative charges, and other experimental factors.
Control
Pancreas Lysate (NB820-59244)
COS-7 Lysate (NB800-PC26)
Bone Marrow Lysate (NB820-59283)
HeLa Lysate (NBP2-36450)
HepG2 Lysate (NBP2-36451)
HeLa Lysate (NBP2-36452)
HepG2 Lysate (NBP2-36453)
HIF-1 alpha Lysate (NBP2-64982)
Control Peptide
HIF-1 alpha Recombinant Protein (NBC1-18422)
Reviewed Applications
Read 21 Reviews rated 4.4
using
NB100-479 in the following applications:

Publications
Read Publications using
NB100-479 in the following applications:

Reactivity Notes

Hamster reactivity reported in scientific literature (PMID: 24997364 and 24997360). Fish reactivity reported in scientific literature (PMID: 29203887).

Packaging, Storage & Formulations

Storage
Store at 4C. Do not freeze.
Buffer
PBS
Preservative
0.05% Sodium Azide
Concentration
1.0 mg/ml
Purity
Immunogen affinity purified

Alternate Names for HIF-1 alpha Antibody

  • ARNT-interacting protein
  • Basic-helix-loop-helix-PAS protein MOP1
  • BHLHE78
  • Class E basic helix-loop-helix protein 78
  • HIF 1A
  • HIF1 alpha
  • HIF-1 alpha
  • HIF1A
  • HIF-1a
  • HIF-1-alpha
  • HIF1-alpha
  • hypoxia inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor)
  • hypoxia-inducible factor 1-alpha
  • Member of PAS protein 1
  • member of PAS superfamily 1
  • MOP1HIF1-ALPHA
  • PAS domain-containing protein 8
  • PASD8alpha subunit (basic helix-loop-helix transcription factor)

Background

HIF1 (hypoxia-inducible factor 1), a heterodimeric transcription factor complex central to cellular response to hypoxia, consists of two subunits (HIF-1 alpha and HIF-1 beta) which are basic helix-loop-helix proteins of the PAS (Per, ARNT, Sim) family. Expression of HIF-1 alpha protein is regulated by cellular oxygen level alterations as well as in oxygen-independent manner via different cytokines (through the PI3K-AKT-mTOR pathway), growth factors, oncogenic activation, or loss of tumor suppressor function etc. In normoxic cells, HIF-1 alpha is proline hydroxylated leading to a conformational change that promotes its binding to the VHL (von Hippel Lindau) protein E3 ligase complex; ubiquitination and followed by rapid proteasomal degradation. Hypoxia as well as chemical hydroxylase inhibitors (desferrioxamine, cobalt etc.) inhibit HIF-1 alpha degradation and lead to its accumulation in the cells, whereas, contrastingly, HIF-1 beta/ARNT (AhR nuclear translocator) remains stable under both conditions. Besides their critical role in hypoxic response, HIF1s regulates the transcription of genes responsible for angiogenesis, erythropoiesis/iron-metabolism, glucose metabolism, cell proliferation/survival, adipogenesis, carotid body formation, B lymphocyte development and immune reactions.

Limitations

This product is for research use only and is not approved for use in humans or in clinical diagnosis. Primary Antibodies are guaranteed for 1 year from date of receipt.

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Publications for HIF-1 alpha Antibody (NB100-479)(278)

We have publications tested in 8 confirmed species: Human, Mouse, Rat, Canine, Fish, Hamster, Moues, Rabbit.

We have publications tested in 11 applications: ChIP, FLOW, IB, ICC/IF, IHC, IHC-Fr, IHC-P, IHC-WhMt, IP, WB, WB,ChIP.


Filter By Application
ChIP
(3)
FLOW
(2)
IB
(1)
ICC/IF
(23)
IHC
(22)
IHC-Fr
(5)
IHC-P
(28)
IHC-WhMt
(1)
IP
(6)
WB
(160)
WB,ChIP
(1)
All Applications
Filter By Species
Human
(87)
Mouse
(106)
Rat
(17)
Canine
(1)
Fish
(1)
Hamster
(2)
Moues
(1)
Rabbit
(1)
All Species
Showing Publications 1 - 10 of 278. Show All 278 Publications.
Publications using NB100-479 Applications Species
Tawari EP. Investigation of chemoresistant mechanisms in triple negative breast. Thesis. Mar 1 2016 (WB, ICC/IF, Human) WB, ICC/IF Human
Pandya PH, Wilkes DS. HIF-1alpha regulates CD55 expression in airway epithelium. Thesis. 2015 Sep 01 (IHC-P, WB, Mouse, Human) IHC-P, WB Mouse, Human
Aoki A, Nakashima A, Kusabiraki T et al. Trophoblast-Specific Conditional Atg7 Knockout Mice Develop Gestational Hypertension Am. J. Pathol. Aug 27 2018 [PMID: 30165042] (IHC-P, Mouse) IHC-P Mouse
Burman A, Kropski JA, Calvi CL et al. Localized hypoxia links ER stress to lung fibrosis through induction of C/EBP homologous protein JCI Insight Aug 23 2018 [PMID: 30135303] (WB, Mouse) WB Mouse
Logue SE, McGrath EP, Cleary P et al. Inhibition of IRE1 RNase activity modulates the tumor cell secretome and enhances response to chemotherapy Nat Commun Aug 15 2018 [PMID: 30111846] (WB, Human) WB Human
Ow CPC, Ngo JP, Ullah MM et al. Absence of renal hypoxia in the subacute phase of severe renal ischemia reperfusion injury Am. J. Physiol. Renal Physiol. Aug 15 2018 [PMID: 30110566] (WB, Rat) WB Rat
Tong B, Pantazopoulou V, Johansson E et al. The p75 neurotrophin receptor enhances HIF-dependent signaling in glioma Exp. Cell Res. Aug 6 2018 [PMID: 30092219] (ICC/IF, Mouse) ICC/IF Mouse
Wu JC, Tsai HE, Liu GS et al. Autophagic cell death participates in POMC-induced melanoma suppression. Cell Death Discov. Jul 10 2018 [PMID: 30062060] (WB, Human) WB Human
Regan Anderson TM, Ma S, Perez kerkvliet C et al. Taxol Induces Brk-dependent Prosurvival Phenotypes in TNBC Cells through an AhR/GR/HIF-driven Signaling Axis. Mol. Cancer Res. Jul 10 2018 [PMID: 29991529] (WB, Human) WB Human
Dai Z, Zhu MM, Peng Y et al. Therapeutic Targeting of Vascular Remodeling and Right Heart Failure in PAH with HIF-2alpha Inhibitor Am. J. Respir. Crit. Care Med. Jun 20 2018 [PMID: 29924941] (WB, Human) WB Human
Show All 278 Publications.

Reviews for HIF-1 alpha Antibody (NB100-479) (21) 4.421

Average Rating: 4.4
(Based on 21 reviews)
We have 21 reviews tested in 3 species: Human, Mouse, Rat.

Reviews using NB100-479:
Filter by Applications
WB
(16)
IHC-P
(4)
ChIP
(1)
All Applications
Filter by Species
Human
(10)
Mouse
(9)
Rat
(2)
All Species
Images Ratings Applications Species Date Details
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Human 09/18/2018
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Summary

ApplicationWestern Blot
Sample TestedHuman Cancer Cells
SpeciesHuman
LotAN-1
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Anonymous
IHC-P Mouse 10/25/2017
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedIHC on parrafin sections (mouse)
SpeciesMouse
LotAK-4
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Kristina Cook
WB Human 02/23/2017
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Summary

ApplicationWestern Blot
Sample TestedU251 glioma cell line; whole cell lysate
SpeciesHuman

Comments

Comments4 hour hypoxia (0.5% oxygen) incubation
 
reviewed by:
Anonymous
WB Mouse 09/21/2016
View

Summary

ApplicationWestern Blot
Sample TestedMouse kidney
SpeciesMouse

Blocking

Blocking DetailsBlocked with 5% Nonfat Dry Milk in 1x TBS-T, 1 hour at room temperature

Primary Anitbody

Dilution RatioDilution - 5% BSA in TBS-T solution buffer

Secondary Antibody

Secondary DescriptionHorse Anti-mouse IgG, HRP-linked Antibody
Secondary Manufacturer Cat##7076
Secondary Concentration2ul in 10ml of 5% Milk in TBS-T

Details

Detection NotesChemiluminescent detection
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Mouse 08/06/2016
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Summary

ApplicationWestern Blot
SpeciesMouse
FileView PDF
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Zhongxiao Wang
WB Mouse 10/05/2015
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Summary

ApplicationWestern Blot
Sample Testedmouse retina
SpeciesMouse
LotNB100-479

Blocking

Blocking Details5%milk/T-BST buffer, room temparature, 2hr

Primary Anitbody

Dilution Ratio1:1000 in 5%milk/T-BST buffer, 4 degree, overnight

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP
Secondary Manufacturer Cat#Sigma AQ132P
Secondary Concentration1:3000

Details

Detection NotesDevelop machine, 30s exposure shows clear bands

Comments

CommentsThere's a non-specific band above for mouse retina samples
 
reviewed by:
Anonymous
WB Human 08/23/2015
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Summary

ApplicationWestern Blot
Sample TestedHuman cancer cell whole cell lysate
SpeciesHuman

Blocking

Blocking DetailsBlocking with 5% non-fat milk for 30 min

Primary Anitbody

Dilution Ratio1:1000, overnight

Secondary Antibody

Secondary DescriptionDonkey anti-rabbit IgG, HRP
Secondary Manufacturer Cat#GE Healthcare, NA934V
Secondary Concentration1:2000

Details

Detection NotesRegular Protocol
 
reviewed by:
Anonymous
WB Human 03/27/2015
View

Summary

ApplicationWestern Blot
Sample TestedHela lysates treated with MG132
SpeciesHuman

Blocking

Blocking Details5% milk in TBST

Primary Anitbody

Dilution Ratio1:2000

Secondary Antibody

Secondary DescriptionGoat anti-rabbit

Details

Detection Notesusing the regular pico from pierece, detection by Bio-rad imager, 1 min exposure time, DMSO as the NC, MG132 as the PC
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Anonymous
WB Mouse 02/18/2015
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Summary

ApplicationWestern Blot
Sample TestedMouse heart
SpeciesMouse
LotJ1

Blocking

Blocking Details5% milk in TBST for 1h at RT

Primary Anitbody

Dilution Ratio1:1000 in 5% milk TBST overnight at 4°C

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP-coupled, 1:10 000 in 5% milk TBST, 1h at RT
Secondary Manufacturer Cat#AffiniPure Goat Anti-Rabbit IgG (H+L) from Jackson Immunolab
Secondary Concentration1:10 000

Details

Detection NotesECL Clarity, exposure time 30s

Comments

Comments- The signal was strong, it might be useful to decrease primary antibody concentration to reduce background noise.
 
reviewed by:
Anonymous
ChIP Human 12/15/2014
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Summary

ApplicationChromatin Immunoprecipitation
Sample TestedHEK293
SpeciesHuman
LotAA-1

Primary Anitbody

Dilution RatioUse 5ug antibody per 1ml lysate, and incubate at 4oC overnight

Details

Detection NotesReal-time PCR.
Fixation/Cross-LinkingElute the beads with 1% SDS, 0.1M NaHCO3 and then reverse the cross-linking by incubating at 65oC for 6 hours.
DNS SharingCovaris sonicator, 6 seconds for 200-500bp
Immunoprecipitation DetailsMillipore protein G magnetic beads 50ul/1ml lysate at room temperature for 10 minutes.
Crosslinking/Reversal Details1% formaldehyde for 5 minutes at room temperature
DNA Prurification DetailsQiagen PCR cleanup kit.
 
reviewed by:
Anonymous
WB Mouse 12/12/2014
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Summary

ApplicationWestern Blot
Sample TestedSee PMID 23232625
SpeciesMouse

Blocking

Blocking DetailsSee PMID 23232625

Details

Detection NotesSee PMID 23232625

Comments

CommentsThis antibody against HIF-1alpha reproducibly recognizes a single band in western blots assessing protein lysates from hypoxia-treated primary mouse aortic endothelial cells.Published in PMID: 23232625.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Xiuquan Luo
WB Human 09/12/2014
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Summary

ApplicationWestern Blot
Sample TestedHEK293 cell lysate
SpeciesHuman
CommentsAntibody detects some non-specific bands, but give a good HIF1a signal at indicated MW.

Blocking

Blocking Details1xPBST with 5% non-fat milk, RT 1 hour

Primary Anitbody

Dilution Ratio1xPBST with 5% non-fat milk, overnight 4C, 1:1000 dilution

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG-HRP
Secondary Manufacturer Cat#sc-2004
Secondary Concentration1:2000 dilution

Details

Detection NotesECL dura, exposured for 2-5 min

Comments

CommentsAntibody detects some non-specific bands, but give a good HIF1a signal at indicated MW.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Ming Lu
WB Human 08/15/2014
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Summary

ApplicationWestern Blot
Sample TestedHuman Pancreatic Cancer Cell line
SpeciesHuman
LotAA
CommentsVery clean antibody, but it has multiple bands.

Blocking

Blocking Details2% cold fish geletin in TBS buffer, room temperature for 1hour

Primary Anitbody

Dilution Ratio1:1000 overnight at 4C in blocking buffer

Secondary Antibody

Secondary DescriptionGoat anti-rabbit IgG, HRP
Secondary Manufacturer Cat#Cell Signaling, #7074
Secondary Concentration1:5000

Details

Detection Notes1 min exposure shows band at correct MW.

Comments

CommentsVery clean antibody, but it has multiple bands.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Molly Jenkins
WB Human 07/18/2014
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Summary

ApplicationWestern Blot
Sample TestedNuclear fraction, Human cell line
SpeciesHuman

Blocking

Blocking Details5% milk in TBST, 1hr at RT

Primary Anitbody

Dilution Ratio1:1000

Secondary Antibody

Secondary DescriptionGoat anti-rabbit HRP
Secondary Concentration1:20,000
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Jason Lee
WB Human 10/29/2013
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Summary

ApplicationWestern Blot
Sample TestedHuman MCF7 breast epithelial cells
SpeciesHuman
LotLot X-1
Commentsnuclear extract resolved in 8% SDS-PAGE gel

Blocking

Blocking Details3% BSA in TBST buffer 1 hour room temp

Primary Anitbody

Dilution Ratio1:2000 dilution in 3% BSA TBST 1 hour room temp

Secondary Antibody

Secondary Descriptiongoat anti-rabbit IgG HRP
Secondary Manufacturer Cat#Sigma
Secondary Concentration1:5000 in 5% skim milk TBST

Details

Detection NotesLAS4000 2 second exposure

Comments

Commentsnuclear extract resolved in 8% SDS-PAGE gel
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Anonymous
IHC-P Mouse 06/27/2013
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample Testedparaffin section of skin wound
SpeciesMouse
CommentsAntibody works much better when you find the necessary dilution which is quite low. Works best around 1:5000 to 1:7000.

Primary Anitbody

Dilution Ratio1:6000, room tempereature in Dako antibody diluent, overnight, 4oC

Secondary Antibody

Secondary Descriptionanti rabbit HRP polymer, Dako

Details

Fixation Details4% paraformaldehyde, DAKO heat mediated antigen retrieval, 20 mins, 98 deg C
Wash DescriptionDako wash buffer, Tris, contains Tween 20

Comments

CommentsAntibody works much better when you find the necessary dilution which is quite low. Works best around 1:5000 to 1:7000.
Western Blot HIF-1 alpha NB100-479
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reviewed by:
Sandhya Khurana
WB Rat 02/05/2012
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Summary

ApplicationWestern Blot
Sample TestedPC12 nuclear extract, Sample Amount: 50ug
SpeciesRat

Blocking

Blocking DetailsBlocking Buffer: 5% Milk in TBST, Blocking Time: 30 minutes, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000, Incubation Dilution Buffer: 5% Milk/TBST, Incubation Time: overnight, Incubation Temp: 4 C

Secondary Antibody

Secondary DescriptionSecondary Ab Dilution Ratio: 1:2000

Details

Detection NotesMethod:ECL, Time:2min, PosCtrl: Lysate from cells overexpressing HIF1a, NegCtrl: Untreated cells, Specific Bands:110-120kDa
 
reviewed by:
Anonymous
WB Rat 01/03/2012
View

Summary

ApplicationWestern Blot
Sample TestedPC12 cell whole lysate, Sample Amount: 25ug
SpeciesRat
LotP1
CommentsThis antibody detected HIF-1a.

Blocking

Blocking DetailsBlocking Buffer: 5% Milk in TBST, Blocking Time: 1 hour, Blocking Temp: Room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000, Incubation Dilution Buffer: blocking buffer, Incubation Time: overnight, Incubation Temp: 4 C

Comments

CommentsThis antibody detected HIF-1a.
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Anonymous
IHC-P Mouse 07/30/2011
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample Testednormal mouse kidney
SpeciesMouse
CommentsNuclear staining of (active) Hif-1alpha + cytoplasmic staining of (inactive) Hif-1alpha in normal mouse kidney (formalin-fixed, paraffin-embedded). No staining detected in controls. Staining done according to CSAII kit instructions. In addition, a permeabilization step was included (20 min. PBS + 0.5% Triton X-100) prior to Protein Block. Detection Method: DAB, Detection Time: 10 minutes, Positive Control: normal mouse kidney, Negative Control: rabbit isotype, antibody pre-absorbed with Hif-1alpha protein (NBC1-18422), Deparaffinization: 2x5 min. xylene, Antigen Retrieval: Target Retrieval Solution (Dako), 25 min. in pressure cooker

Blocking

Blocking DetailsBlocking Buffer: Protein Block (CSAII kit, Dako), Blocking Time: 15 minutes, Blocking Temp: room temperature

Primary Anitbody

Dilution RatioDilution Ratio: 1:20,000, Incubation Dilution Buffer:Ab Diluent, background reducing (Dako), Incubation Time/Temp:overnight @ 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: CSAII Rabbit Link (Dako), Secondary Ab Dilution: 1:1
Secondary Manufacturer Cat#K1501

Details

Detection NotesPlease see Comments
Fixation DetailsPreparation: perfused PBS-formalin, postfixed overnight @ 4C in formalin, paraffin-embedded, Deparaffinization: 2x5 min. xylene

Comments

CommentsNuclear staining of (active) Hif-1alpha + cytoplasmic staining of (inactive) Hif-1alpha in normal mouse kidney (formalin-fixed, paraffin-embedded). No staining detected in controls. Staining done according to CSAII kit instructions. In addition, a permeabilization step was included (20 min. PBS + 0.5% Triton X-100) prior to Protein Block. Detection Method: DAB, Detection Time: 10 minutes, Positive Control: normal mouse kidney, Negative Control: rabbit isotype, antibody pre-absorbed with Hif-1alpha protein (NBC1-18422), Deparaffinization: 2x5 min. xylene, Antigen Retrieval: Target Retrieval Solution (Dako), 25 min. in pressure cooker
Immunohistochemistry-Paraffin HIF-1 alpha NB100-479
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reviewed by:
Paramahamsa Maturu
IHC-P Human 03/01/2011
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Summary

ApplicationImmunohistochemistry-Paraffin
Sample TestedHuman Kidney tumor
SpeciesHuman
LotI
CommentsThat works great without any background staining in the negative control. Detection Method: ABC and DAB, Positive Control: Glioblastoma, Negative Control: Without primary Ab, Deparaffinization: Xylen and ethanol, Antigen Retrieval: Tris -EDTA pH 9.0, 45 minutes at 95 degrees Celsius in water bath

Blocking

Blocking DetailsBlocking Buffer:.10% Normal Goat Serum in 1XPBS, Blocking Time: 1 hour, Blocking Temp: RT in humidified chamber

Primary Anitbody

Dilution RatioDilution Ratio: 1:100, Incubation Dilution Buffer: 2.5% Normal Goat Serum in 1X PBS, Incubation Time/Temp: overnight at 4C

Secondary Antibody

Secondary DescriptionSecondary Ab: Biotinylated Anti Rabbit IgG (H+L) made in Goat, Secondary Ab Dilution: 1:500
Secondary Manufacturer Cat#BA-1000

Details

Detection NotesPlease see Comments
Fixation DetailsSample Preparation: Paraffin embedded, Deparaffinization: Xylen and ethanol

Comments

CommentsThat works great without any background staining in the negative control. Detection Method: ABC and DAB, Positive Control: Glioblastoma, Negative Control: Without primary Ab, Deparaffinization: Xylen and ethanol, Antigen Retrieval: Tris -EDTA pH 9.0, 45 minutes at 95 degrees Celsius in water bath
Western Blot HIF-1 alpha NB100-479
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reviewed by:
JORCYK LAB
WB Mouse 02/01/2011
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Summary

ApplicationWestern Blot
Sample Tested4T1 cells whole cell lysate
SpeciesMouse
CommentsThe Hif1a band shows up even if the lysate concentration is very low. There are some background bands, but is acceptable, and there is quite a bit of lot to lot variations with this product... unfortunately.

Blocking

Blocking DetailsBlocking Buffer: 5% milk or 3% BSA, Blocking Time: overnight, Blocking Temp: 4 degrees Celsius

Primary Anitbody

Dilution RatioDilution Ratio: 1:1000-1:5000, Incubation Dilution Buffer: TBS-T or PBS-T, Incubation Time: 1 hr-overnight, Incubation Temp: 4C

Secondary Antibody

Secondary DescriptionSecondary Ab Dilution Ratio: 1:10,000-1:150,000

Details

Detection NotesMethod: ECL, Exposure Time: 10sec-10min, Pos.Ctrl: ECL reactive ladder, Bands:~120kDa(specific), ~80,55,72kDa(non-specific)

Comments

CommentsThe Hif1a band shows up even if the lysate concentration is very low. There are some background bands, but is acceptable, and there is quite a bit of lot to lot variations with this product... unfortunately.

Product General Protocols

Video Protocols

WB Video Protocol
ChIP Video Protocol
ChIP Webinar
ICC/IF Video Protocol

FAQs for HIF-1 alpha Antibody (NB100-479). (Showing 1 - 9 of 9 FAQs).

  1. Why is there a difference between the theoretical MW for HIF1A and the observed MW for HIF-1 alpha?
    • HIF1A, like many other proteins, has post-translational modifications. Depending on the size, amount and nature of the post-translational modifications, it can cause subtle to very large changes in molecular weight.
  2. Which antibody(ies) do you recommend for the detection of HIF-1a by immunohistochemistry in the sections of paraffin-embedded mouse liver samples? I would appreciate if you can give me several choices and rank them in the order of performance. My goal is to distinguish HIF upregulation by prolyl hydroxylase inhibitor in different liver cells.
    • All of our antibodies are of high quality and are well tested/validated in species/applications we list on the datasheet. However, as desired by you, I am suggesting you four HIF-1 alpha antibodies based upon customer reviews, as well as the number of peer reviewed publications in which these products has been cited by researchers from reputed institutes. (1) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-105) (cited in at least 218 peer reviewed publications) (2) HIF-1 alpha Antibody (cat# NB100-479) (cited in at least 51 peer reviewed publications) (3) HIF-1 alpha Antibody (H1alpha67) (cat# NB100-123 ) (cited in at least 38 peer reviewed publications) (4) HIF-1 alpha Antibody (cat# NB100-449 ) (cited in at least 31 peer reviewed publications).
  3. I would like to know, does it exist path for detection HIF 1 in the venous blood before and after revascularization of the leg?
    • We are not entirely sure if HIF-1 alpha will be present in the leg after revascularization. It may be present but you may want to search the literature to see if this has been looked at before. If not, then this would certainly be an experiment worth doing.
  4. What is the molecular weight (kDa) of protein HIF 1 alpha in western blot?
    • The theoretical molecular weight of HIF 1-alpha is ~93kDa, however, you will likely see a band between 100-120kDa due to phosphorylation.
  5. Do you sell a positive and negative control for the HIF-1 alpha antibody (NB100-479)?
    • For Western blot, the best positive control that we have is a CoCl2-treated Cos-7 cell lysate, catalog # NB800-PC26. The kit comes with a negative control as well. If you are doing ICC, then you could treat your cells with CoCl2 or expose them to hypoxic conditions.
  6. What do think the blocking buffer should be?
    • We recommend 5% non-fat dry milk in TBS.
  7. We need to examine tissue-specific knockout of HIF-1a in mouse lungs. We have generated smooth muscle specific knockout of HIF-1a, and would like to demonstrate the knockdown in lung tissue sections by IHC or immunofluorescence. The problem is that all the antibodies for the mouse seem to have too much non-specific staining. Can you recommend an antibody that would be good for mouse lung tissue IHC or immunofluorescence?
    • The antibody with Catalog # NB100-479 is one of our best sellers and has 5 star rating in customer reviews. Please view the product datasheet to see the publications in which researchers have cited this product, over 10 of which are involved in mouse models.
  8. Is there a WB image for this antibody?
    • We do have a WB image for the antibody base project. It looks like they ordered an HRP conjugated format, and that specific format page does not have the image. We do all of our testing on the base product.
  9. Will this antibody detect HIF-1 alpha protein by WB on a breast cancer cell line in normal condition (without hypoxia)?
    • If the breast cancer gene turned on the HIF1 alpha gene, there should be some expression of the protein, even without hypoxic induction. However, this expression may be very low, and depending on the length of time it took to lyse and/or fix the cells, may have degraded in the presence of oxygen. This may also be affected by the presence or lack of any HIF stabilizers in the lysate buffer. We talk about these stabilizers and other testing protocols in our white paper here: http://www.novusbio.com/whitepapers/cellular-response-hypoxia.html. I would recommend trying to detect HIF expression in cancer cell lines that you run both positive and negative controls along side the cell lines to ensure that any bands detected are in fact HIF1 alpha.

Control Lysate(s)

Secondary Antibodies

 

Isotype Controls

Other Available Formats

Alexa Fluor 405 Labeled NB100-479AF405
Alexa Fluor 488 Labeled NB100-479AF488
Alexa Fluor 647 Labeled NB100-479AF647
Alexa Fluor 700 Labeled NB100-479AF700
Biotin Labeled NB100-479B
DyLight 405 Labeled NB100-479V
DyLight 488 Labeled NB100-479G
DyLight 550 Labeled NB100-479R
DyLight 650 Labeled NB100-479C
FITC Labeled NB100-479F
HRP Labeled NB100-479H

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Blogs on HIF-1 alpha. Showing 1-10 of 30 blog posts - Show all blog posts.

Bad news for stomach cancer: BAMBI protein inhibits gastric carcinoma via TGF-beta/epithelial-mesenchymal transition signaling
By Jamshed Arslan Pharm.D. Gastric carcinoma is the second leading cause of cancer-related deaths worldwide. One of the key features of gastric carcinoma is acidosis, which promotes growth and metastasis of gastric...  Read full blog post.

Developmental regulator Daam2 promotes glial cell tumors by degrading Von Hippel-Lindau protein
By Jamshed Arslan Pharm.D. Glioblastoma is an aggressive type of cancer that forms from the star-shaped glial cells of the central nervous system, called astrocytes. Intriguingly, several genes linked to glioblasto...  Read full blog post.

Stemness for Surviving Hypoxia: TGF-beta/Smad Signaling in Multiple Myeloma
By Jamshed Arslan Pharm.D. Multiple myeloma (MM) is a cancer of antibody-producing plasma cells. The bone marrow (BM) of MM patients is hypoxic, and MM cells overexpress many cancerous genes that are regulated by hy...  Read full blog post.

Forecasting and Targeting a Rare Cancer with Hypoxia-Inducible Factor
By Jamshed Arslan Pharm.D. Cancers of nerve, adipose, and other soft tissues are called soft tissue sarcomas (STS). Malignant peripheral nerve sheath tumor (MPNST) is an example of a rare and hard-to-treat STS; eve...  Read full blog post.

The role of HIF-1 Alpha signaling in the retina under hypoxic conditions
Hypoxia inducible factor 1 (HIF-1) is a protein that plays an essential role in hypoxia, or low levels of cellular oxygen. HIF-1 is a heterodimeric protein that consists of a constitutively expressed beta subunit and oxygen related alpha subunit. ...  Read full blog post.

The relationship between Ki67 and HIF-1 in cancer
Ki67, also known as MKI67, is best known as the leading marker of cellular proliferation. Ki67 is regulated by a balance between synthesis and degradation, and often carries a very short half-life.  First discovered to be located to dividing cells,...  Read full blog post.

Controls for HIF-1 Alpha's WB, ICC-IF, IHC, IP & FLOW Analysis
Tips on positive and negative controls for HIF-1 alpha antibodies is one of the most Frequently Asked Questions on Hypoxia and HIFs. Here are top 5 suggestions from Novus Biologicals: The degradation of HIF1 alpha is the most common issue whic...  Read full blog post.

Understanding the relationship between HIF-1 alpha, Hypoxia and Epithelial-Mesenchymal Transition
Epithelial-mesenchymal transition (EMT) is a natural process by which epithelial cells lose their polarity and intercellular adhesion, and gain the migratory invasive properties of mesenchymal stem cells that can differentiate into a variety of cel...  Read full blog post.

HIF-2 alpha: HIF1A's Homologue with Similar and Divergent Functions
HIF-2 alpha is a member of the heterodimeric hypoxia-inducible factors/HIFs family (HIF-1, HIF-2, and HIF-3) which contains a common beta subunit but differ in their alpha subunits. Also called as EPAS1 or Mop2, HIF-2 alpha regulates cellular adapt...  Read full blog post.

HIF-3 alpha: a versatile target with hypoxia dependent and independent functions
By: Subhash GangarHIF-3 alpha (hypoxia-inducible factor 3-alpha/ HIF3A) represents an isoform of HIF-alpha subunits which heterodimerize with stable beta subunit (HIF-beta) for the regulation of HIF target genes through binding to hypoxia respon...  Read full blog post.

Showing 1-10 of 30 blog posts - Show all blog posts.

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Recent Reviews

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Anonymous
09/18/2018
Application: WB
Species: Human

Anonymous
10/25/2017
Application: IHC-P
Species: Mouse

Kristina Cook
02/23/2017
Application: WB
Species: Human

Bioinformatics

Gene Symbol HIF1A
Entrez
Uniprot