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ARNT/HIF-1 beta Antibody

Catalog # NB100-110 | Novus Biologicals a Bio-Techne Brand
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NB100-110
NB100-110SS

Key Product Details

Species Reactivity

Human, Mouse, Rat, Bovine, Canine, Ferret, Fish, Sheep

Applications

Chromatin Immunoprecipitation (ChIP), Gel Supershift Assay, Immunoblotting, Immunocytochemistry/ Immunofluorescence, Immunohistochemistry, Immunohistochemistry-Paraffin, Immunoprecipitation, Simple Western, Western Blot

Label

Unconjugated

Antibody Source

Polyclonal Rabbit IgG

Concentration

This product is unpurified. The exact concentration of antibody is not quantifiable.

Product Summary for ARNT/HIF-1 beta Antibody

Immunogen

ARNT/HIF-1 beta Antibody was developed against a fusion protein to human HIF-1 beta containing amino acids 496-789. [UniProt# P27540]

Reactivity Notes

ARNT/HIF-1 beta antibody shows Fish reactivity as reported in scientific literature (PMID: 30334616). Canine reactivity reported in scientific literature (PMID:32885302)

Localization

Nuclear

Specificity

ARNT/HIF-1 beta Antibody is specific for HIF-1 beta/ARNT. It is not known if NB100-110 cross-reacts with ARNT2 which is related to HIF-1 beta/ARNT but is the product of a different gene.

Clonality

Polyclonal

Host

Rabbit

Isotype

IgG

Theoretical MW

86.6 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.

Scientific Data Images for ARNT/HIF-1 beta Antibody

Chromatin Immunoprecipitation: ARNT/HIF-1 beta Antibody [NB100-110] - ChIP analysis with ARNT/HIF-1 beta antibody [NB100-110]. Allele specific HIF-binding at the rs12814794 associated enhancer. B) Allele-specific qPCR for rs12814794 from DNA fragments isolated in HIF-ChIP experiments (HIF-1A and HIF-1B) or input DNA from primary renal tubular cells (PTC, n = 4 individuals). DNA from individuals homozygous for the AA or GG genotype were used as positive controls. C) Quantification of the two different alleles (A or G) at rs12814794 in the ChIP-seq reads from HIF-1A and HIF-1B immunoprecipitations (PTC #1 Fig 3). Image collected and cropped by CiteAb from the following publication (//doi.org/10.1371/journal.pgen.1006872) licensed under a CC-BY license.
Western Blot: ARNT/HIF-1 beta Antibody [NB100-110] - Analysis of HIF-1 beta using ARNT/HIF-1 beta antibody [NB100-110] in MCF7 whole cell lysate. Theoretical molecular weight is 86.6 kDa. Observed molecular weight ~95 kDa.
Immunohistochemistry: ARNT/HIF-1 beta Antibody [NB100-110] - Immunohistochemical staining of human skin epidermis with ARNT/HIF-1 beta antibody [NB100-110].

Applications for ARNT/HIF-1 beta Antibody

Application
Recommended Usage

Chromatin Immunoprecipitation (ChIP)

1:10-1:500

Immunohistochemistry

1:150

Immunohistochemistry-Paraffin

1:150

Immunoprecipitation

1:10-1:500

Simple Western

1:1000

Western Blot

1:2000
Please Note: Optimal dilutions of this antibody should be experimentally determined.

Reviewed Applications

Read 3 reviews rated 5 using NB100-110 in the following applications:

Published Applications

Read 70 publications using NB100-110 in the following applications:

Formulation, Preparation, and Storage

Purification

Unpurified

Formulation

Whole antisera

Preservative

0.02% Sodium Azide

Concentration

This product is unpurified. The exact concentration of antibody is not quantifiable.

Shipping

The product is shipped with polar packs. Upon receipt, store it immediately at the temperature recommended below.

Stability & Storage

Aliquot and store at -20C or -80C. Avoid freeze-thaw cycles.

Background: ARNT/HIF-1 beta

Aryl hydrocarbon nuclear translocator (ARNT), also commonly known as Hypoxia-inducible factor 1-beta (HIF-1 beta), is a ubiquitously expressed transcription factor that is part of the basic-helix-loop-helix (bHLH)-Per-ARNT-Sim (PAS) family (1, 2). Human arnt is located on chromosome 1q21 and encodes a protein 789 amino acids (aa) in length with a theoretical molecular weight of 87 kDa (1). Structurally, ARNT has a DNA binding bHLH domain, two PAS domains required for dimerization, and a transactivation domain/PAC region (1). ARNT belongs to the Class II bHLH-PAS proteins and is able to homodimerize or heterodimerize with the Class I proteins including AHA, AHRR, HIF-1 alpha, HIF-2 alpha, NPAS1, and SIM1 (2). Dimerization allows for efficient DNA binding and regulation of their target genes (2).

ARNT has an important role in two specific signaling pathways - the aryl hydrocarbon receptor (AhR) and the hypoxia inducible factor (HIF) pathway (1). In the AhR pathway, AhR in the cytosol is typically inactive and bound to heat shock protein 90 (hsp90) (3). Upon activation and ligand binding by environmental pollutants such as dioxins, AhR is translocated to the nucleus, dissociates from hsp90, and dimerizes with ARNT, leading to binding to response elements and expression of target genes including monooxygenases (1, 3). In the HIF pathway, under hypoxia (low oxygen) conditions prolylhydroxylase domain (PHD) enzymes and factor inhibiting HIF (FIH) are inhibited. HIF-1 alpha (or HIF-2 alpha) accumulates and is transported to the nucleus where it heterodimerizes with ARNT, allowing for binding to target gene's hypoxia response element (HRE), recruitment of coactivators, and transcription (1, 3). HIF-induced gene transcription plays a large role in tumor progression by promoting invasion, metastasis, de-differentiation and altered metabolism, and angiogenesis (1). While HIF-1 alpha's stability is dependent upon oxygen conditions, HIF-1 beta is stable in both normoxia and hypoxia (1-3).

The bHLH-PAS family and ARNT have been linked with a variety of pathologies and diseases including cancer, metabolic diseases, autoimmune diseases, and psychiatric disorders (2). ARNT/AHR is expressed in the skin and its pathway activation enhances skin barrier function and epidermal terminal differentiation, thus AHR agonists are currently being used as therapeutics for atopic dermatitis and psoriasis (4). Accordingly, studies of Arnt-deficient mice show profound abnormalities in skin barrier function and keratinization (4). Additionally, studies suggest that ARNT plays an important role in diabetes and beta-cell function (5). Islets from patients with type 2 diabetes have a significantly decreased ARNT expression compared to glucose-tolerant control donors (5). Modulation and stimulation of the HIF pathway may be a potential therapeutic strategy for treating type 2 diabetes and metabolic syndrome (5).

Alternate names for ARNT/HIF-1 beta include aryl hydrocarbon receptor nuclear translocator, BHLHE2, class E basic helix-loop-helix protein 2, Dixon receptor nuclear translocator, Hypoxia-inducible factor 1-beta, nuclear translocator, and TANGO.

References

1. Mandl, M., & Depping, R. (2014). Hypoxia-inducible aryl hydrocarbon receptor nuclear translocator (ARNT) (HIF-1beta): is it a rare exception?. Molecular medicine (Cambridge, Mass.). https://doi.org/10.2119/molmed.2014.00032

2. Wu, D., & Rastinejad, F. (2017). Structural characterization of mammalian bHLH-PAS transcription factors. Current opinion in structural biology. https://doi.org/10.1016/j.sbi.2016.09.011

3. Esser, C., & Rannug, A. (2015). The aryl hydrocarbon receptor in barrier organ physiology, immunology, and toxicology. Pharmacological reviews.https://doi.org/10.1124/pr.114.009001

4. Furue, M., Hashimoto-Hachiya, A., & Tsuji, G. (2019). Aryl Hydrocarbon Receptor in Atopic Dermatitis and Psoriasis. International journal of molecular sciences. https://doi.org/10.3390/ijms20215424

5. Girgis, C. M., Cheng, K., Scott, C. H., & Gunton, J. E. (2012). Novel links between HIFs, type 2 diabetes, and metabolic syndrome. Trends in endocrinology and metabolism: TEM, https://doi.org/10.1016/j.tem.2012.05.003

Long Name

Aryl Hydrocarbon Receptor Nuclear Translocator

Alternate Names

HIF-1 beta, HIF1 beta, TANGO

Entrez Gene IDs

405 (Human); 11863 (Mouse); 25242 (Rat)

Gene Symbol

ARNT

UniProt

Product Documents for ARNT/HIF-1 beta Antibody

Certificate of Analysis

To download a Certificate of Analysis, please enter a lot number in the search box below.

Product Specific Notices for ARNT/HIF-1 beta Antibody

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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Protocols

View specific protocols for ARNT/HIF-1 beta Antibody (NB100-110):

[[URL:https://www.novusbio.com/products/arnt-hif-1-beta-antibody_nb100-110#pr… beta Antibody]]


Western Blot Procedure

1. Resolve aliquots (15 mg) of induced * nuclear protein extracts on a SDS/6% polyacrylamide gel.

2. Transfer to nitrocellulose membranes in 20 mM Tris-HCl (pH 8.0)/150 mM glycine/20% (vol/vol) methanol.

3. Block membranes for 1.5 hours with 1X western wash buffer containing 5% non-fat dry milk (NFDM).

4. Incubate membranes for 1.5 hours at room temperature (RT) in NB100-110 diluted 1:2,000 ** in 1X western wash/5% NFDM.

5. Wash with 1X western wash for 35 minutes at RT (1 X 15 minutes, 2 X 10 minutes).

6. Incubate membranes with HRP conjugated anti-Rabbit IgG for 1 hour (RT) in 1X western wash/5% NFDM. Wash with 1X western wash for 35 minutes at RT (1 X 15 minutes, 2 X 10 minutes).

7. Drain membrane and place on saran wrap.

8. Using Amersham ECL Kit, mix equal volumes of two reagents. Pour over membrane (protein side facing up). Let solution sit on membrane for 15-20 seconds.

9. Drain membrane and place on new saran wrap

10. Wrap up membrane and expose to film.

11. Develop accordingly.

Notes: If hypoxia treatment is not hypoxic enough (less than 2% oxygen to get an induction), signal will be absent. Also, if the harvest time is too slow or there are not enough protease inhibitors, etc., the induced protein will be rapidly lost as HIF-1beta has a very short half-life. Whole cell extracts or nuclear extracts of hypoxia induced cell lines (293, Hep3B, COS7, Hepa) are useful as a positive control. Nuclear Extract Preparation Reference: Wang and Semenza. "Purification and Characterization of Hypoxia-Inducible Factor 1". Journal of Biological Chemistry. 270(3): 1230-1237, 1995.


IHC-FFPE sections

I. Deparaffinization:
A. Treat slides with Xylene: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.
B. Treat slides with 100% Reagent Alcohol: 3 changes for 5 minutes each. Drain slides for 10 seconds between changes.

II. Quench Endogenous Peroxidase:
A. Place slides in peroxidase quenching solution: 15-30 minutes.
To Prepare 200 ml of Quenching Solution:
Add 3 ml of 30% Hydrogen Peroxide to 200 ml of Methanol.
Use within 4 hours of preparation
B. Place slides in distilled water: 2 changes for 2 minutes each.

III. Retrieve Epitopes:
A. Preheat Citrate Buffer. Place 200 ml of Citrate Buffer Working Solution into container, cover and place into steamer. Heat to 90-96 degrees Celcius.
B. Place rack of slides into hot Citrate Buffer for 20 minutes. Cover.
C. Carefully remove container with slides from steamer and cool on bench, uncovered, for 20 minutes.
D. Slowly add distilled water to further cool for 5 minutes.
E. Rinse slides with distilled water. 2 changes for 2 minutes each.

IV. Immunostaining Procedure:
A. Remove each slide from rack and circle tissue section with a hydrophobic barrier pen (e.g. Liquid Blocker-Super Pap Pen).
B. Flood slide with Wash Solution. Do not allow tissue sections to dry for the rest of the procedure.
C. Drain wash solution and apply 4 drops of Blocking Reagent to each slide and incubate for 15 minutes.
D. Drain Blocking Reagent (do not wash off the Blocking Reagent), apply 200 ul of Primary Antibody solution to each slide, and incubate for 1 hour.
E. Wash slides with Wash Solution: 3 changes for 5 minutes each.
F. Drain wash solution, apply 4 drops of Secondary antibody to each slide and incubate for 1 hour.
G. Wash slides with Wash Solution: 3 changes for 5 minutes each.
H. Drain wash solution, apply 4 drops of DAB Substrate to each slide and develop for 5-10 minutes. Check development with microscope.
I. Wash slides with Wash Solution: 3 changes for 5 minutes each.
J. Drain wash solution, apply 4 drops of Hematoxylin to each slide and stain for 1-3 minutes. Increase time if darker counterstaining is desired.
K. Wash slides with Wash Solution: 2-3 changes for 2 minutes each.
L. Drain wash solution and apply 4 drops of Bluing Solution to each slide for 1-2 minutes.
M. Rinse slides in distilled water.
N. Soak slides in 70% reagent alcohol: 3 minutes with intermittent agitation.
O. Soak slides in 95% reagent alcohol: 2 changes for 3 minutes each with intermittent agitation.
P. Soak slides in 100% reagent alcohol: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
Q. Soak slides in Xylene: 3 changes for 3 minutes each with intermittent agitation. Drain slides for 10 seconds between each change.
R. Apply 2-3 drops of non-aqueous mounting media to each slide and mount coverslip.
S. Lay slides on a flat surface to dry prior to viewing under microscope.


NOTES:
-Use treated slides (e.g. HistoBond) to assure adherence of FFPE sections to slide.
-Prior to deparaffinization, heat slides overnight in a 60 degrees Celcius oven.
-All steps in which Xylene is used should be performed in a fume hood.
-For Epitope Retrieval, a microwave or pressure cooker may be substituted for the steamer method. Adjust times as necessary depending on conditions.

-For the initial IHC run with a new primary antibody, test tissues with and without Epitope Retrieval. In some instances, Epitope Retrieval may not be necessary.

-200 ul is the recommended maximum volume to apply to a slide for full coverage. Using more than 200 ul may allow solutions to wick off the slide and create drying artifacts. For small tissue sections less than 200 ul may be used.

-5 minutes of development with DAB Substrate should be sufficient. Do not develop for more than 10 minutes. If 5 minutes of development causes background staining, further dilution of the primary antibody may be necessary.

-Hematoxylin should produce a light nuclear counterstain so as not to obscure the DAB staining. Counterstain for 1-1 1/2 minutes for nuclear antigens. Counterstain for 2-3 minutes for cytoplasmic and membranous antigens. If darker counterstaining is desired increase time (up to 10 minutes).

FAQs for ARNT/HIF-1 beta Antibody

Showing  1 - 2 of 2 FAQs Showing All
  • Q: So this NB100-110 does not have a concentration. How much would you recommend to use per chip reaction where I normally use 5 to 10 ug of antibody?

    A: Our HIF-1 beta product NB100-110 is provided as whole antisera, so we have not determined the protein concentration. We recommend using a 1:10-1:500 dilution for ChIP. You will need to determine the optimal amount for your experiment.

  • Q: Do you have a concentration for NB100-110?  what dilution do you recommend for a ChIP

    A: NB100-110 is actually unpurified product, so there is no concentration listed. The recommended dilution for chromatin Immunoprecipitation for this product is 1:10-1:500.

Showing  1 - 2 of 2 FAQs Showing All
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