Overview

  • Product name
  • Description
    Rabbit polyclonal to PHD3
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IP, ICC/IF, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 50 - 150 of Human PHD3/prolyl hydroxylase.

    Read Abcam's proprietary immunogen policy (Peptide available as ab30781.)

  • Positive control
    • In WB this antibody gave a positive signal in whole cell, nuclear and cytoplasmic extracts of DFO-treated HeLa cells. In IF this antibody gave a positive signal in the following formaldehyde fixed cell line: HepG2.

Properties

Applications

Our Abpromise guarantee covers the use of ab30782 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
IP Use at an assay dependent concentration.
ICC/IF Use a concentration of 5 µg/ml.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 27 kDa (predicted molecular weight: 27 kDa).

Target

  • Function
    Catalyzes the post-translational formation of 4-hydroxyproline in hypoxia-inducible factor (HIF) alpha proteins. Hydroxylates HIF-1 alpha at 'Pro-564', and HIF-2 alpha. Functions as a cellular oxygen sensor and, under normoxic conditions, targets HIF through the hydroxylation for proteasomal degradation via the von Hippel-Lindau ubiquitination complex. May play a role in cell growth regulation in muscle cells and in apoptosis in neuronal tissue. Promotes cell death through a caspase-dependent mechanism.
  • Tissue specificity
    Widely expressed at low levels. Expressed at higher levels in heart (cardiac myocytes, aortic endothelial cells and coronary artery smooth muscle) and placenta.
  • Sequence similarities
    Contains 1 Fe2OG dioxygenase domain.
  • Cellular localization
    Cytoplasm. Nucleus.
  • Information by UniProt
  • Database links
  • Alternative names
    • Egl 9 family hypoxia inducible factor 3 antibody
    • Egl nine homolog 3 (C. elegans) antibody
    • Egl nine homolog 3 antibody
    • Egl nine like protein 3 isoform antibody
    • EGL9 homolog of C. elegans 3 antibody
    • EGLN3 antibody
    • EGLN3_HUMAN antibody
    • Factor responsive smooth muscle protein antibody
    • HIF Prolyl Hydroxylase 3 antibody
    • HIF-PH3 antibody
    • HIF-prolyl hydroxylase 3 antibody
    • HIFP4H3 antibody
    • HIFPH3 antibody
    • HPH-1 antibody
    • HPH-3 antibody
    • Hypoxia-inducible factor prolyl hydroxylase 3 antibody
    • P4H3 antibody
    • PHD3 antibody
    • Prolyl Hydroxylase Domain Containing Protein 3 antibody
    • Prolyl hydroxylase domain-containing protein 3 antibody
    • SM20 antibody
    see all

Images

  • All lanes : Anti-PHD3 antibody (ab30782) at 1 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)
    Lane 3 : Human HeLa Nuclear DFO treated
    Lane 4 : Human HeLa Cytoplasmic DFO treated

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 27 kDa
    Observed band size: 27 kDa
    Additional bands at: 18 kDa (possible non-specific binding)


    Exposure time: 20 minutes


    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 5% Bovine Serum Albumin before being incubated with ab30782 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.

  • All lanes : Anti-PHD3 antibody (ab30782) at 1/1000 dilution

    Lane 1 : Mouse embroyonic fibroblast cell lysate. Treated 21% O2 for 24 hours
    Lane 2 : Mouse embroyonic fibroblast cell lysate. Treated 1% O2 for 24 hours

    Lysates/proteins at 5 µg per lane.

    Secondary
    All lanes : Goat polyclonal anti-rabbit HRP-conjugate at 1/10000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 27 kDa


    Exposure time: 30 seconds

    See Abreview

  • ab30782 immunoprecipitating PHD3 from rat PC12 (whole cell lysate - the cells were either normoxia (21% oxygen) or hypoxia (1% hypoxia). A protein A matrix was used with the antibody at a dilution of 1/1000. A non-Abcam antibody was used in the subsequent western blot. The doublet observed is specific to PHD3 - most probably a modified form or an alternately spliced form. Note: This antibody IP's a hypoxia inducible species of ~27 kDa and ~30 kDa. This is the same molecular weight as the human and mouse isoform, not the SM-20 variant with the N-terminal mitochondrial leader sequence.

    Lane 1: normoxia - input

    Lane 2: hypoxia - input

    Lane 3: normoxia - control antibody IP

    Lane 4: hypoxia - control antibody IP

    Lane 5: normoxia - ab30782 IP

    Lane 6: hypoxia - ab30782 IP

    See Abreview

  • ICC/IF image of ab30782 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab30782 at 5µg/ml overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References

This product has been referenced in:
  • Li D  et al. Armadillo repeat containing 12 promotes neuroblastoma progression through interaction with retinoblastoma binding protein 4. Nat Commun 9:2829 (2018). Read more (PubMed: 30026490) »
  • Ognibene M  et al. Immunohistochemical analysis of PDK1, PHD3 and HIF-1a expression defines the hypoxic status of neuroblastoma tumors. PLoS One 12:e0187206 (2017). Read more (PubMed: 29117193) »
See all 8 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (mouse embryonic fibroblast)
Loading amount
5 µg
Specification
mouse embryonic fibroblast
Treatment
21% O2 and 1% O2 for 24hrs
Gel Running Conditions
Reduced Denaturing (10%)
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 25°C

Abcam user community

Verified customer

Submitted May 17 2013

Answer

Thank you for contacting us.

As always, if we do not advise a certain method on our datasheet, the customer can use the one which they usually use within their lab. This is the case of ab110025.

For ab65979 I would suggest checking the publication which is cited within the application as we added IHC-P due to this application and haven't tested the antibody ourselves. Nonetheless, it is covered by our guarantee.

In the case of ab30782 we suggest a heat mediated antigen retrieval. The image description on the datasheet mentions that citrate buffer was used, but if the customer prefers to use EDTA it should be fine as well.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

Read More

Answer

Thank you for confirming this information and for your help and cooperation with this case.

As requested, I have asked our accounting department to issue you with a credit note. This can then be redeemed against the invoice of a future order.

Credit ID: ####

As usual if you have any further questions regarding this credit note, please contact the accounts department by email at creditcontrol@abcam.com. Please refer to the credit ID number in any correspondence with the accounting department.

I would like to wish the customer good luck with their research. The technical team is always at your service, should you require further expert advice.

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Question

Name: Dawinbio Dawinbio
Product code: 30782
Lot number: GR26814-1
Inquiry: Non-specific band I transfected siRNA against PHD3 #1,#2, and #3 . Three days later, siRNA was verified by RT-PCR and western blot with abcam antibody. RT-PCR data showed successful knockdown of PHD3 (see attached figure)but western blot with Ab30782 did not show any reduced PHD3. Recently we got our own custom ab against PHD3 which detected endogenous PHD3 and exactly matched with RT-PCR data.

1) Abcam product code ab
Ab30782
2) Abcam order reference number or product batch number
GR26814-1,
2-1) storage temperature of antibody
-20oC
3) Description of the problem
Non specific detection of PHD3
4) Sample preparation:
Type of sample and species(whole cell lysates, fraction, recombinant protein…and Human,
mouse…): whole cell lysate, 1% SDS, TRis buffer, endogenous PHD3, human 786-O cell
line
Lysis buffer : 1% SDS, Tris buffer
Protease inhibitors:Complete mini
Phosphatase inhibitors : sigma co.
Reducing agent DTT
Product name Anti-PHD3 ab
Cat no Ab30782 Batch no GR26814-1
구매시기 Mar, 2012 구매수량 1
Boiling for ≥5 min? Yes
Protein loaded ug/lane or cells/lane 25 ug
Positive control: siRNA of PHD3 transfected
Negative control : non transfected cell lysate
5) Percentage of gel 10% SDS-PAGE
Type of membrane NC
Protein transfer verified Prestained molecular marker
Blocking agent and concentration 5% non-fat dried milk in TBST
Blocking time One and half hour
Blocking temperature at Room temperature
6) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1:1000
Diluent buffer 1% non-fat dried milk in TBST
Incubation time overnight
Incubation temperature: 4°C
7) Secondary antibody:
Species:goat
Isotype:
Reacts against: rabbit IgG
Concentration or dilution 1:10000
Diluent buffer 5% non-fat dried milk in TBST
Incubation time: one hour
Incubation temperature: room temperature
Fluorochrome or enzyme conjugate:HRP
8) Washing after primary and secondary antibodies:
Buffer: TBST
Number of washes three times, 5 min each
9)Detection method: Thermo scientific, SuperSignal West Pico
10) How many times have you run this staining? two
-Do you obtain the same results every time? yes
-Have you run a "No Primary" control? no
-What steps have you altered to try and optimize the use of this antibody? siRNA , shRNA tried
Document attachment: Attaching images of your blot is strongly recommended and can greatly
speed up our investigation of your problem.
I transfected siRNA against PHD3 #1,#2, and #3 . Three days later, siRNA was verified by
RT-PCR and western blot with abcam antibody.
RT-PCR data showed successful knockdown of PHD3 (see attached figure)but western
blot with Ab30782 did not show any reduced PHD3.
Recently we got our own custom ab against PHD3 which detected endogenous PHD3 and exactly matched with RT-PCR data.

Read More
Answer

Thank you for taking the time to complete our questionnaire and contact us. I am sorry to hearthe customer has had difficulty obtaining satisfactory results from this antibody.

The details you have kindlysent will provide us with vital information for our monitoring of product quality. I would like to reassure you that this antibody is tested and covered by our guarantee for WB and human samples.

I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been successful. Reviewing the details, I am sorry there very few tips to provide on this occasion to help improve the results. I can suggest you have regrettably received a bad vial.

I am pleased to offer you a free of charge replacement or credit note in compensation.

Thank you for your cooperation. I look forward to hearing from you with details of how you would like to proceed.

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Immunoprecipitation
Sample
Rat Cell lysate - whole cell (PC12)
Total protein in input
500 µg
Specification
PC12
Treatment
Normoxia and Hypoxia (1% Oxygen, 4 hrs)
Immuno-precipitation step
Protein A

Abcam user community

Verified customer

Submitted Oct 30 2007

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Rat Cell lysate - whole cell (PC12)
Loading amount
40 µg
Specification
PC12
Treatment
Normoxia and Hypoxia (1% Oxygen, 4 hrs)
Gel Running Conditions
Reduced Denaturing
Blocking step
Milk as blocking agent for 45 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Jun 20 2007

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"

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