Overview

  • Product name
    Anti-P4HB antibody - ER Marker
    See all P4HB primary antibodies
  • Description
    Rabbit polyclonal to P4HB - ER Marker
  • Host species
    Rabbit
  • Tested applications
    Suitable for: IHC-P, WB, IP, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Human
    Predicted to work with: Chicken, Cow
  • Immunogen

    Synthetic peptide corresponding to Human P4HB aa 400-500 conjugated to keyhole limpet haemocyanin.
    Database link: P07237
    (Peptide available as ab31810)

  • Positive control
    • This antibody gave a positive signal in the lysates: Human Brain Whole Tissue Lysate, Human Kidney Whole Tissue Lysate, Human Liver Whole Tissue Lysate and Human Testes Whole Tissue Lysate, Mouse Liver Whole Tissue Lysate; Rat Liver Whole Tissue Lysate.

Properties

Applications

Our Abpromise guarantee covers the use of ab31811 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
IHC-P 1/800. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 57 kDa (predicted molecular weight: 57 kDa).Can be blocked with Human P4HB peptide (ab31810).
IP Use a concentration of 5 µg/ml.
ICC/IF Use a concentration of 1 µg/ml.

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

  • P4HB (PDIA1) was immunoprecipitated using 0.5mg Mouse Pancreas tissue lysate, 5µg of Rabbit polyclonal to and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).

    The antibody was incubated under agitation with Protein G beads for 10min, Mouse Pancreas tissue lysate lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab31811.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 57kDa, non specific - as present in control (lane 2); 55kDa: We are confident this was due to slight lane contamination and the band seen in the IP lane is our target of interest; P4HB (PDIA1)

  • All lanes : Anti-P4HB antibody - ER Marker (ab31811) at 1 µg/ml

    Lane 1 : Human Brain Whole Tissue Lysate
    Lane 2 : Human Kidney Whole Tissue Lysate at 20 µg
    Lane 3 : Human Liver Whole Tissue Lysate at 20 µg
    Lane 4 : Human Testes Whole Tissue Lysate at 20 µg

    Secondary
    All lanes : IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution

    Performed under reducing conditions.

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



    Several smaller bands are seen in Human Liver Whole Tissue Lysate. These are predicted to be break-down products of P4HB (PDIA1); the banding pattern is consistent with that observed in Liver lysates for other available P4HB (PDIA1) antibodies.

  • Image courtesy of Human Protein Atlas

    ab31811 staining P4HB (PDIA1 in Human gall bladder. The paraffin embedded tissue was incubated with ab31811 (1/800 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6. ab31811 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines.

    Further results for this antibody can be found at www.proteinatlas.org

  • ICC/IF image of ab31811 stained human HEK 293 cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab31811, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue). This antibody also gave a positive IF result in HeLa and MCF7 cells.

     

  • All lanes : Anti-P4HB antibody - ER Marker (ab31811) at 1 µg/ml

    Lane 1 : Liver (Mouse) Tissue Lysate
    Lane 2 : Liver (Rat) Tissue Lysate

    Lysates/proteins at 10 µg per lane.

    Secondary
    All lanes : Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

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


    Exposure time: 30 seconds
  • IHC image of ab31811 staining in pancreas formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab31811, 1µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

References

This product has been referenced in:
  • Doerflinger SY  et al. Membrane alterations induced by nonstructural proteins of human norovirus. PLoS Pathog 13:e1006705 (2017). Read more (PubMed: 29077760) »
  • Guiraud S  et al. HANAC Col4a1 Mutation in Mice Leads to Skeletal Muscle Alterations due to a Primary Vascular Defect. Am J Pathol 187:505-516 (2017). Read more (PubMed: 28056338) »
See all 7 Publications for this product

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Nematode Tissue lysate - whole (nematode)
Gel Running Conditions
Reduced Denaturing (12% gel)
Loading amount
30 µg
Specification
nematode
Blocking step
Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C

Jianlong Zhao

Verified customer

Submitted Jun 15 2018

Answer

Sorry for the dely in getting back to you. I now have further information concerning Anti-PDI antibody (ab31811). The human brain tissue lysate used for the Western blot presented on the datasheet of ab31811 was taken from the parietal lobe. The protocol used to perform this Western blot is as follows: 1. Tissue lysate prepared 2. Lysate (20-40 µg) loaded on SD-page gel and transferred to Nitrocellulose membrane  3. Membrane blocked using 5% BSA in 0.1% TBST, for 1 hour at room temperature 4. Primary diluted to 1 µg/ml in 5% BSA TBST, incubated with the membrane overnight at 4°C with gentle rocking 5. Was step performed: 0.1% TBST 2 brief rinses followed by 1x15 min then 3x10 min at room temperature. 6. Secondary antibody incubated with membrane: IR Dye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/15000 dilution in 5% BSA 0.1% TBST, at room temperature for 1 hour, with gentle rocking. 7. Repetition of wash step 5 I hope this information has been of help. If you need any further information or help in ordering please do not hesitate to contact me again.

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Answer

Thank you for contacting us again. When I said that ab31811 had not been charaterised as well as ab2792 I simply meant that this antibody had not been used and referenced in as many publications and had not had as many reviews by our other customers. This does not mean that it will perform any differently. I think ab31811 would be suitable for the experiment you will be performing. I am currently investigating exactly which type of brain sample was used with ab31811 to obtain the blot presented on the datasheet and as soon as I have the answer I will get back to you. You are correct in thinking that if you have problems obtaining the results expected with Western blotting of human samples you would be entitled to a refund. We usually deal with such cases (which are quite rare) by initially looking into the experiment you have been performing by getting you to fill out a brief questionnaire (5-10 min max) in order to understand the problem and investigate the case internally if need be, then if we can't find anything to help you otimise the protocol we would offer a free of charge replacement or a refund, whichever you prefer. We are very flexible and understand that you know what you are doing so therefore usually the questionnaire is just to establish if we need to initiate some additional testing on our side. We do not try to withhold refunds. I hope this information has been of help, if you need any further information please do not hesitate to ask. I will let you know once I have received confirmation of the obtain sample used and the protocol used.

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Answer

Thank you for contacting us. I have looked into the products that we have available for the species and application you are interested in (Western blotting with human brain samples) for PDI and Connexin. 1. PDI There are two antibodies which look like they would be promising for your research: ab2792 is a mouse monoclonal which has been used in the following publications to perform Western blotting and immunoprecipitation, although not with the same tissue samples you are hoping to analyse: Western blotting with human granulocytes, antibody used as loading control: Petheo GL et al. Molecular and functional characterization of Hv1 proton channel in human granulocytes. PLoS One 5:e14081 (2010). WB; Human. PubMed: 21124855 Immunoprecipitaiton illustrating the association of PDI with TAP in M553 (melanoma cell line): Rizvi SM & Raghavan M Mechanisms of Function of Tapasin, a Critical Major Histocompatibility Complex Class I Assembly Factor. Traffic 3, 332-347, (2009). IP; Human. PubMed: PubMed: 20070606 This antibody has also been used with several human cell lines to perform Western blotting with quite clean results as well as one report of using Human Tissue lysate - whole (Brain): https://www.abcam.com/index.html?datasheet=2792&tab=abreviews&intabreviewid=13102 Alternatively, as discussed yesterday, whilst ab31811 (rabbit polyclonal) has not been as well charaterised as ab2792, it has been used to perform Western blotting with Human brain whole tissue lysate in house and I can obtain a detailed protocol of how this is performed if you are still interested in this antibody. Both of these antibodies have been tested for human samples and Western blotting and as such would be covered by our Abpromise guarantee, more information of which can be found from the link below. This means that if you have any problems obtaining the results you would expect with this antibody with your human brain samples, and we are unable to help you obtain better results, you would be entitled to an alternative antibody or a refund. www.abcam.com/abpromise 2. Connexin As there are so many different forms of connexin, in order to suggest the best antibody for you could you please specify which form you are wanting to study? We have antibodies against: Connexin 36/GJA9, 30.3/GJB4, 40.1/GJD4, 59/GJA10, 26/GJB2, 30/GJB6, 62GJA10, 46/GJA3, 31/GJB3, 50/GJA8, 23/GJE1, 31.1/GJB5, 47GJC2, 45/GJC1 I hope this information has been of help. If you'd like more help in selecting a suitable anti-connexin antobody, or would like any other information please do let me know.

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

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