EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

Novus Biologicals | Catalog # NBP1-30440

Novus Biologicals

Key Product Details

Validated by

Biological Validation

Species Reactivity

Validated:

Human, Rat

Cited:

Human, Rat, Ovine

Predicted:

Bovine (98%), Mouse (98%), Primate (99%). Backed by our 100% Guarantee.

Applications

Validated:

Immunohistochemistry, Immunohistochemistry-Paraffin, Western Blot, Simple Western

Cited:

Immunohistochemistry-Paraffin, Western Blot, IF/IHC

Label

Unconjugated

Antibody Source

Monoclonal Mouse IgG1 Clone # EG188e/d5

Format

BSA Free
View all EGLN3/PHD3 Primary Antibodies »

Product Specifications

Immunogen

Partial recombinant human PHD3/HIF Prolyl Hydroxylase 3 (UniProt# Q9H6Z9)

Localization

Cytoplasm. Nucleus.

Clonality

Monoclonal

Host

Mouse

Isotype

IgG1

Theoretical MW

27 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 EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

Western Blot: EGLN3/PHD3 Antibody (EG188e/d5)BSA Free [NBP1-30440]

Western Blot: EGLN3/PHD3 Antibody (EG188e/d5)BSA Free [NBP1-30440]

Western Blot: EGLN3/PHD3 Antibody (EG188e/d5) [NBP1-30440] - PHD3 expression in SK-N-BE(2) whole cell lysates. Cells were treated in normoxia (left) and in hypoxia (right). Image from verified customer review.
Immunohistochemistry: EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free [NBP1-30440]

Immunohistochemistry: EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free [NBP1-30440]

Immunohistochemistry: EGLN3/PHD3 Antibody (EG188e/d5) [NBP1-30440] - Analysis of HIF Prolyl Hydroxylase 3 in human renal cancer using DAB with hematoxylin counterstain.
Western Blot: EGLN3/PHD3 Antibody (EG188e/d5)BSA Free [NBP1-30440]

Western Blot: EGLN3/PHD3 Antibody (EG188e/d5)BSA Free [NBP1-30440]

Western Blot: EGLN3/PHD3 Antibody (EG188e/d5) [NBP1-30440] - Analysis of HIF Prolyl Hydroxylase 3 in (1) Cobalt chloride treated COS-7 nuclear extracts and (2) Untreated COS-7 nuclear extracts.
Simple Western: EGLN3/PHD3 Antibody (EG188e/d5)BSA Free [NBP1-30440]

Simple Western: EGLN3/PHD3 Antibody (EG188e/d5)BSA Free [NBP1-30440]

Simple Western: EGLN3/PHD3 Antibody (EG188e/d5) [NBP1-30440] - Lane view shows a specific band for PHD3/HIF Prolyl Hydroxylase 3 in 0.5 mg/ml of Hypoxic HeLa lysate. This experiment was performed under reducing conditions using the 12-230 kDa separation system.

Applications for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

Application
Recommended Usage

Immunohistochemistry

1:400

Immunohistochemistry-Paraffin

1:400

Simple Western

1:1000

Western Blot

10 ug/ml
Application Notes

This HIF Prolyl Hydroxylase 3 antibody is useful for Immunohistochemistry paraffin embedded sections and Western blot, where a band is seen approx. 27 kDa. Prior to immunostaining paraffin tissues, antigen retrieval with sodium citrate buffer (pH 6.0) is recommended.

In Simple Western only 10 - 15 uL of the recommended dilution is used per data point.
See Simple Western Antibody Database for Simple Western validation: Tested in Hypoxic HeLa lysate 0.5 mg/mL, separated by Size, antibody dilution of 1:1000, apparent MW was 32 kDa. Separated by Size-Wes, Sally Sue/Peggy Sue.

Reviewed Applications

Read 4 reviews rated 4.8 using NBP1-30440 in the following applications:

Formulation, Preparation, and Storage

Purification

Protein G purified

Formulation

PBS

Format

BSA Free

Preservative

0.05% Sodium Azide

Concentration

1 mg/ml

Shipping

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

Stability & Storage

Store at 4C short term. Aliquot and store at -20C long term. Avoid freeze-thaw cycles.

Background: EGLN3/PHD3

HIF prolyl 4-hydroxylases (PHDs) are proyl hydroxylase domain-containing enzymes (PHD1/ Egln2, PHD2/ Egln1, PHD3/ Egln3, and P4H-TM) which are known for their role in mediating physiological responses to hypoxic stress via modulation of HIF1alpha expression levels. HIF-alpha subunit is regulated by hydroxylation, both by a family of PHDs leading to ubiquitination and proteasomal degradation, and by transcriptional inactivation following asparaginyl hydroxylation by FIH (factor inhibiting HIF). When oxygen levels are normal, HIF Prolyl Hydroxylase 3 (HIF-PH3/ PHD3/ Egln3) hydroxylates a specific proline found in HIF1A'a NODD/CODD domains and also hydroxylates HIF2A with preference for CODD site for HIF1A/HIF2A. Once hydroxylated, HIFs undergo proteasomal degradation via von Hippel-Lindau (VHL) ubiquitination complex. Upon hypoxic trigger, the hydroxylation reaction is tempered which let HIFs to escape degradation process leading to their nuclear translocation, heterodimerization with HIF1B, and increased expression of hypoxy-inducible genes. HIF-PH3 is the most important isozyme in limiting physiological activation of HIFs (particularly HIF2A) in hypoxia. Besides HIFs, in hypoxic conditions HIF-PH3 also hydroxylates PKM (thereby limiting glycolysis) and hydroxylates/regulates the stability of ADRB2. In cardiomyocytes, HIF-PH3 inhibits Bcl2's anti-apoptotic effect by disrupting the BAX-BCL2 complex, and in neurons, HIF-PH3 has a NGF-induced proapoptotic effect mediated via CASP3 activity regulation. HIF-PH3 also plays a crucial role in DNA damage response by hydroxylating TELO2, promoting its interaction with ATR which is required for activation of the ATR/CHK1/p53 pathway.

Long Name

Egl Nine Homolog 3/Prolyl Hydroxylase Domain-containing Protein 3

Alternate Names

HIFPH3, PHD3

Entrez Gene IDs

112399 (Human)

Gene Symbol

EGLN3

UniProt

Q9H6Z9

Additional EGLN3/PHD3 Products

Product Documents for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

Certificate of Analysis

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

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Product Specific Notices for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

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.

Citations for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

Customer Reviews for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free (4)

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Customer Images

Showing  1 - 4 of 4 reviews Showing All
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  • EGLN3/PHD3 Antibody (EG188e/d5)
    Name: Anonymous
    Application: Western Blot
    Sample Tested: HBE-16
    Species: Human
    Verified Customer | Posted 04/16/2019
    PHD3
    EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free NBP1-30440
  • Name: Anonymous
    Application: Western Blot
    Sample Tested: MCF-7 whole cell lysate
    Species: Human
    Verified Customer | Posted 11/20/2015
    EGLN3 in MCF7 cells
    EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free NBP1-30440
  • EGLN3/PHD3 Antibody (EG188e/d5)
    Name: Anonymous
    Application: Western Blot
    Sample Tested: SK-N-BE(2) whole cell lysate
    Species: Other
    Verified Customer | Posted 10/30/2014
  • EGLN3/PHD3 Antibody (EG188e/d5)
    Name: Anonymous
    Application: Western Blot
    Sample Tested: PC12 whole cell lysate
    Species: Other
    Verified Customer | Posted 08/27/2014

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Protocols

View specific protocols for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free (NBP1-30440):


Immunohistochemistry-Paraffin Embedded Sections

Antigen Unmasking:
Bring slides to a boil in 10 mM sodium citrate buffer (pH 6.0) then maintain at a sub-boiling temperature for 10 minutes. Cool slides on bench-top for 30 minutes.

Staining:
1. Wash sections in deionized water three times for 5 minutes each.
2. Wash sections in wash buffer for 5 minutes.
3. Block each section with 100-400 ul blocking solution for 1 hour at room temperature.
4. Remove blocking solution and add 100-400 ul diluted primary antibody. Incubate overnight at 4C.
5. Remove antibody solution and wash sections in wash buffer three times for 5 minutes each.
6. Add 100-400 ul biotinylated diluted secondary antibody. Incubate 30 minutes at room temperature.
7. Remove secondary antibody solution and wash sections three times with wash buffer for 5 minutes each.
8. Add 100-400 ul Streptavidin-HRP reagent to each section and incubate for 30 minutes at room temperature.
9. Wash sections three times in wash buffer for 5 minutes each.
10. Add 100-400 ul DAB substrate to each section and monitor staining closely.
11. As soon as the sections develop, immerse slides in deionized water.
12. Counterstain sections in hematoxylin.
13. Wash sections in deionized water two times for 5 minutes each.
14. Dehydrate sections.
15. Mount coverslips.


Western Blot Protocol

1. Perform SDS-PAGE (4-12% MOPS) on samples to be analyzed, loading 40 ug of total protein per lane.
2. Transfer proteins to Nitrocellulose according to the instructions provided by the manufacturer of the transfer apparatus.
3. Rinse membrane with dH2O and then stain the blot using Ponceau S for 1-2 minutes to access the transfer of proteins onto the nitrocellulose membrane. Rinse the blot in water to remove excess stain and mark the lane locations and locations of molecular weight markers using a pencil.
4. Rinse the blot in TBS for approximately 5 minutes.
5. Block the membrane using 5% NFDM + 1% BSA in TBS + Tween, 1 hour at RT.
6. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
7. Dilute the mouse anti-PHD3 primary antibody (NBP1-30328) in blocking buffer and incubate 1 hour at room temperature.
8. Rinse the membrane in dH2O and then wash the membrane in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each.
9. Apply the diluted mouse-IgG HRP-conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer [TBS + 0.1% Tween] 3 times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions (Pierce ECL).

**Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%, provided it does not interfere with antibody-antigen binding.

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FAQs for EGLN3/PHD3 Antibody (EG188e/d5) - BSA Free

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  • Q: I received an inquiry about Anti-PHD3/HIF Prolyl Hydroxylase 3 Antibody (EG188e/d5), #NBP1-30440. Could you let me know if this antibody cross-reacts with PHD1 or PHD2?

    A: We have not tested our PHD3 antibody for cross reactivity with PHD1 or PHD2, therefore we are unaware of any possible cross reactivity.

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Associated Pathways

HIF Enhancer Pathways HIF Enhancer Pathway Thumbnail