Overview

  • Product name
    Anti-P4HB antibody [EPR9499]
    See all P4HB primary antibodies
  • Description
    Rabbit monoclonal [EPR9499] to P4HB
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WB, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human P4HB aa 450 to the C-terminus (C terminal). The exact sequence is proprietary.

  • Positive control
    • WB: HeLa, HepG2, 293T, Raw264.7, PC-12 and A431 cell lysates. ICC/IF: HepG2 and HeLa cells. IHC-P: Human breast carcinoma, human brain, human kidney, mouse liver and rat liver tissues. Flow Cyt: HepG2 and HeLa cells.
  • General notes

     

     

    Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMab® patents.

    We are constantly working hard to ensure we provide our customers with best in class antibodies. As a result of this work we are pleased to now offer this antibody in purified format. We are in the process of updating our datasheets. The purified format is designated 'PUR' on our product labels. If you have any questions regarding this update, please contact our Scientific Support team.

    This product is a recombinant rabbit monoclonal antibody.

Properties

Applications

Our Abpromise guarantee covers the use of ab137110 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
WB 1/1000 - 1/10000. Predicted molecular weight: 57 kDa.
IHC-P 1/1000. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

See IHC antigen retrieval protocols.

For unpurified use at 1/100 - 1/250.

Flow Cyt 1/100 - 1/1000.

ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.

ICC/IF 1/150 - 1/500.

Target

  • Function
    This multifunctional protein catalyzes the formation, breakage and rearrangement of disulfide bonds. At the cell surface, seems to act as a reductase that cleaves disulfide bonds of proteins attached to the cell. May therefore cause structural modifications of exofacial proteins. Inside the cell, seems to form/rearrange disulfide bonds of nascent proteins. At high concentrations, functions as a chaperone that inhibits aggregation of misfolded proteins. At low concentrations, facilitates aggregation (anti-chaperone activity). May be involved with other chaperones in the structural modification of the TG precursor in hormone biogenesis. Also acts a structural subunit of various enzymes such as prolyl 4-hydroxylase and microsomal triacylglycerol transfer protein MTTP.
  • Sequence similarities
    Belongs to the protein disulfide isomerase family.
    Contains 2 thioredoxin domains.
  • Cellular localization
    Endoplasmic reticulum lumen. Melanosome. Cell membrane. Highly abundant. In some cell types, seems to be also secreted or associated with the plasma membrane, where it undergoes constant shedding and replacement from intracellular sources (Probable). Localizes near CD4-enriched regions on lymphoid cell surfaces. Identified by mass spectrometry in melanosome fractions from stage I to stage IV.
  • Information by UniProt
  • Database links
  • Alternative names
    • Cellular thyroid hormone binding protein antibody
    • Cellular thyroid hormone-binding protein antibody
    • Collagen prolyl 4 hydroxylase beta antibody
    • Disulphide Isomerase antibody
    • DSI antibody
    • EC 5.3.4.1 antibody
    • Endoplasmic reticulum resident protein 59 antibody
    • ER protein 59 antibody
    • ERBA2L antibody
    • ERp59 antibody
    • GIT antibody
    • Gltathione insulin transhydrogenase antibody
    • Glutathione insulin transhydrogenase antibody
    • P4HB antibody
    • P4Hbeta antibody
    • p55 antibody
    • PDI antibody
    • PDIA1 antibody
    • PDIA1_HUMAN antibody
    • PDIR antibody
    • PHDB antibody
    • PO4DB antibody
    • PO4HB antibody
    • Procollagen proline 2 oxoglutarate 4 dioxygenase (proline 4 hydroxylase) beta polypeptide (protein disulfide isomerase associated 1) antibody
    • Procollagen proline 2 oxoglutarate 4 dioxygenase beta subunit antibody
    • PROHB antibody
    • Prolyl 4 hydroxylase beta polypeptide antibody
    • Prolyl 4 hydroxylase beta subunit antibody
    • Prolyl 4 hydroxylase subunit beta antibody
    • Prolyl 4-hydroxylase subunit beta antibody
    • Protein disulfide isomerase associated 1 antibody
    • Protein disulfide isomerase, family A, member 1 antibody
    • Protein disulfide isomerase/oxidoreductase antibody
    • Protein disulfide-isomerase antibody
    • Protocollagen hydroxylase antibody
    • Thbp antibody
    • Thyroid hormone binding protein p55 antibody
    • Thyroid hormone binding protein p55 cellular antibody
    • V erb a avian erythroblastic leukemia viral oncogene homolog 2 like antibody
    see all

Images

  • All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1 µg/ml

    Lane 1 : Wild-type HeLa whole cell lysate
    Lane 2 : P4HB knockout HeLa whole cell lysate
    Lane 3 : HEP G2 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 57 kDa



    Lanes 1 - 3: Merged signal (red and green). Green - ab137110 observed at 57 kDa. Red - loading control, ab9484, observed at 37 kDa.

    ab137110 was shown to specifically react with P4HB (Protein disulfide-isomerase) in wild-type HeLa cells as signal was lost in P4HB knockout cells. Wild-type and P4HB knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% milk. Ab137110 and ab9484 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1 μg/ml and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/10000 dilution (purified)

    Lane 1 : HepG2 whole cell lysate
    Lane 2 : HeLa whole cell lysate
    Lane 3 : HEK293 whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 57 kDa
    Observed band size: 57 kDa



    Blocking and dilution buffer: 5% NFDM/TBST
  • All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/10000 dilution (purified)

    Lane 1 : Raw264.7 whole cell lysate
    Lane 2 : PC-12 whole cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

    Predicted band size: 57 kDa
    Observed band size: 57 kDa



    Blocking and dilution buffer: 5% NFDM/TBST
  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human kidney carcinoma tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of mouse liver tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of rat liver tissue labelling P4HB with purified ab137110 at a dilution of 1/1000. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

  • Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling P4HB with purified ab137110 at a dilution of 1/150. Cells were fixed with 100% methanol and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

    Control 1: primary antibody (1/150) and secondary antibody, ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000).

    Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/1000).

  • Flow Cytometry analysis of HeLa cells labelling P4HB with purified ab137110 at a dilution of 1/300 (red). Cells were fixed with 4% paraformaldehyde. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

  • All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/1000 dilution (unpurified)

    Lane 1 : HepG2 cell lysate
    Lane 2 : HeLa cell lysate
    Lane 3 : 293T cell lysate
    Lane 4 : A431 cell lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : HRP-conjugated goat anti-rabbit IgG at 1/2000 dilution

    Developed using the ECL technique.

    Predicted band size: 57 kDa

  • All lanes : Anti-P4HB antibody [EPR9499] (ab137110) at 1/2000 dilution (unpurified)

    Lane 1 : Mouse brain tissue lysate
    Lane 2 : Mouse heart tissue lysate
    Lane 3 : Mouse kidney tissue lysate
    Lane 4 : Mouse spleen tissue lysate

    Predicted band size: 57 kDa

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human breast carcinoma tissue labelling P4HB with unpurified ab137110 at a dilution of 1/100.

  • Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human brain tissue labelling P4HB with unpurified ab137110 at a dilution of 1/100.

  • Immunocytochemistry/Immunofluorescence analysis of HepG2 cells labelling P4HB (green) with unpurified ab137110 at a dilution of 1/250. Cell nuclei are shown in blue.

  • Overlay histogram showing HepG2 cells stained with unpurified ab137110 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab137110, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was a goat anti-rabbit Alexa Fluor® 488 (IgG H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x106 cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HepG2 cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

References

This product has been referenced in:
  • Smith NT  et al. Redox responses are preserved across muscle fibres with differential susceptibility to aging. J Proteomics 177:112-123 (2018). WB ; Mouse . Read more (PubMed: 29438851) »
  • Zhou Y  et al. P4HB knockdown induces human HT29 colon cancer cell apoptosis through the generation of reactive oxygen species and inactivation of STAT3 signaling. Mol Med Rep N/A:N/A (2018). Read more (PubMed: 30431122) »
See all 4 Publications for this product

Customer reviews and Q&As

1-6 of 6 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - whole cell (HUES7 ES cells)
Gel Running Conditions
Reduced Denaturing (12%)
Loading amount
20 µg
Specification
HUES7 ES cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C

Abcam user community

Verified customer

Submitted Apr 14 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (undifferentiated Human ES cells on MEFs)
Permeabilization
Yes - see blocking buffer
Specification
undifferentiated Human ES cells on MEFs
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: 16°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Feb 23 2016

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (HEPG2)
Specification
HEPG2
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: rt°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Nov 18 2015

Application
Western blot
Sample
Human Cell lysate - whole cell (HEPG2)
Gel Running Conditions
Reduced Denaturing (12.5)
Loading amount
20 µg
Specification
HEPG2
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C

Abcam user community

Verified customer

Submitted Nov 13 2015

Application
Western blot
Loading amount
1e+006 cells
Gel Running Conditions
Reduced Denaturing
Sample
Mouse Cell lysate - whole cell (mouse embryonic stem cells)
Specification
mouse embryonic stem cells
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C

Abcam user community

Verified customer

Submitted Jul 11 2014

Application
Immunocytochemistry/ Immunofluorescence
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 16°C
Sample
Mouse Cell (mouse embryonic stem cell)
Specification
mouse embryonic stem cell
Permeabilization
Yes - 0.1% triton X
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Dec 20 2013

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