Overview

  • Product name

    Anti-ENPP2/ATX antibody [5H3]
    See all ENPP2/ATX primary antibodies
  • Description

    Mouse monoclonal [5H3] to ENPP2/ATX
  • Host species

    Mouse
  • Tested applications

    Suitable for: IHC-P, ELISA, WB, ICC/IFmore details
  • Species reactivity

    Reacts with: Mouse, Human
  • Immunogen

    Recombinant full length protein corresponding to Human ENPP2/ATX.

  • Positive control

    • WB: HEK293 whole cell lysate and in the following human tissue lysates: kidney; placenta; ovary; small intestine. IHC: human tonsil paraffin sections. ICC/IF: HeLa cells.
  • General notes

    This antibody clone is manufactured by Abcam.

    If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.

     This product was previously labelled as ENPP2

     

Properties

Applications

Our Abpromise guarantee covers the use of ab77104 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 Use a concentration of 1 µg/ml.
ELISA Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 100 kDa (predicted molecular weight: 99 kDa).
ICC/IF Use at an assay dependent concentration.

Target

  • Function

    Hydrolyzes lysophospholipids to produce lysophosphatidic acid (LPA) in extracellular fluids. Major substrate is lysophosphatidylcholine. Also can act on sphingosylphosphphorylcholine producing sphingosine-1-phosphate, a modulator of cell motility. Can hydrolyze, in vitro, bis-pNPP, to some extent pNP-TMP, and barely ATP. Involved in several motility-related processes such as angiogenesis and neurite outgrowth. Acts as an angiogenic factor by stimulating migration of smooth muscle cells and microtubule formation. Stimulates migration of melanoma cells, probably via a pertussis toxin-sensitive G protein. May have a role in induction of parturition. Possible involvement in cell proliferation and adipose tissue development. Tumor cell motility-stimulating factor.
  • Tissue specificity

    Predominantly expressed in brain, placenta, ovary, and small intestine. Expressed in a number of carcinomas such as hepatocellular and prostate carcinoma, neuroblastoma and non-small-cell lung cancer. Expressed in body fluids such as plasma, cerebral spinal fluid (CSF), saliva, follicular and amniotic fluids. Not detected in leukocytes. Isoform 1 is more highly expressed in peripheral tissues than in the central nervous system (CNS). Adipocytes only express isoform 1. Isoform 3 is more highly expressed in the brain than in peripheral tissues.
  • Sequence similarities

    Belongs to the nucleotide pyrophosphatase/phosphodiesterase family.
    Contains 2 SMB (somatomedin-B) domains.
  • Post-translational
    modifications

    N-glycosylation, but not furin-cleavage, plays a critical role on secretion and on lysoPLD activity.
  • Cellular localization

    Secreted. Secreted by most body fluids including serum and CSF. Also by adipocytes and numerous cancer cells.
  • Information by UniProt
  • Database links

  • Alternative names

    • ATX antibody
    • ATX X antibody
    • Autotaxin antibody
    • Autotaxin t antibody
    • E NPP 2 antibody
    • E-NPP 2 antibody
    • Ectonucleotide pyrophosphatase/phosphodiesterase 2 antibody
    • Ectonucleotide pyrophosphatase/phosphodiesterase family member 2 antibody
    • Enpp2 antibody
    • ENPP2_HUMAN antibody
    • Extracellular lysophospholipase D antibody
    • FLJ26803 antibody
    • LysoPLD antibody
    • NPP2 antibody
    • PD IALPHA antibody
    • PDNP2 antibody
    • Phosphodiesterase I alpha antibody
    • Phosphodiesterase I/nucleotide pyrophosphatase 2 antibody
    • Plasma lysophospholipase D antibody
    see all

Images

  • All lanes : Anti-ENPP2/ATX antibody [5H3] (ab77104) at 10 µg/ml

    Lane 1 : Human kidney tissue lysate - total protein (ab30203)
    Lane 2 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 99 kDa
    Observed band size: 100 kDa
    why is the actual band size different from the predicted?
    Additional bands at: 180 kDa, 250 kDa, 50 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 20 minutes
  • IHC image of ENPP2/ATX staining in Human Tonsil FFPE section, performed on a 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 ab77104, 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

  • ICC/IF image of ab77104 stained HeLa cells. The cells were 100% methanol fixed (5 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 (ab77104, 10µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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.
  • All lanes : Anti-ENPP2/ATX antibody [5H3] (ab77104) at 10 µg/ml

    Lane 1 : Human placenta tissue lysate - total protein (ab29745)
    Lane 2 : Human ovary tissue lysate - total protein (ab30222)
    Lane 3 : Human small intestine tissue lysate - total protein (ab29276)

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Developed using the ECL technique.

    Performed under reducing conditions.

    Predicted band size: 99 kDa
    Observed band size: 110 kDa why is the actual band size different from the predicted?
    Additional bands at: 300 kDa, 40 kDa, 55 kDa. We are unsure as to the identity of these extra bands.


    Exposure time: 20 minutes


    ENPP2/ATX contains three potential glycosylation sites (SwissProt), which might explain its migration at a higher molecular weight than predicted.

References

This product has been referenced in:

  • Zhang G  et al. ATX-LPA axis facilitates estrogen-induced endometrial cancer cell proliferation via MAPK/ERK signaling pathway. Mol Med Rep 17:4245-4252 (2018). Read more (PubMed: 29328374) »
  • Su SC  et al. Autotaxin-lysophosphatidic acid signaling axis mediates tumorigenesis and development of acquired resistance to sunitinib in renal cell carcinoma. Clin Cancer Res 19:6461-72 (2013). IHC-P ; Human . Read more (PubMed: 24122794) »
See all 2 Publications for this product

Customer reviews and Q&As

1-9 of 9 Abreviews or Q&A

Answer


Based on the immunogen for ab77104, the antibody should recognize all isoforms, but we have not specifically tested this. The immunogen is the full length ENPP2 protein.

Read More

Answer

Thank you for contacting Abcam.

I've compiled a list of primary antibodies that have been tested in mouse tissue via western blot. The only ovary-specific antibodies that I found were anti-human (with only 50% homology with the respective mouse protein), so the ovary antibody listed below is not exclusively-specific to ovary but also gives a positive signal in kidney, placenta, and small intestine:

1. Liver: anti-albumin ab135575 (https://www.abcam.com/albumin-antibody-c-terminal-ab135575.html)
2. Brain: anti-neuron specific beta III tubulin ab18207 (https://www.abcam.com/neuron-specific-beta-iii-tubulin-antibody-neuronal-marker-ab18207.html)
3. Kidney: anti-SLC14A2 ab95365 (https://www.abcam.com/slc14a2-antibody-ab95365.html)
4. Testis: anti-histone H2B ab23913 (https://www.abcam.com/histone-h2b-testis-specific-antibody-ab23913.html)
5. Ovary: anti-ENPP2 (also kidney, placenta, small intestine): ab77104 (https://www.abcam.com/enpp2-antibody-5h3-ab77104.html)

The advantage of this combination of antibodies is that you can use the same anti-rabbit secondary since they were all raised in a rabbit host. Our anti-rabbit secondary antibodies are listed in the link below:
https://www.abcam.com/Search?Keywords=anti-rabbit&fReset=1&pt=-1&source=TopSearch

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

Free Rabbit monoclonal antibody with any purchase of a primary antibody, while stocks last! Quote “RABMAB-XBSMG” in your next primary antibody order. For more information, visit the following link:
https://www.abcam.com/index.html?pageconfig=resource&rid=15447

Read More

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement. This will be the same lot as what you received before. We do not have other lots, but we have also not received other complaints about this one, and it is possible the issue was with the single vial.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

Read More

Answer

Thank you for contacting us about this issue. Your protocol appears to be correct, and you should be seeing a signal with 50ug per lane and a 1/400 dilution. Sometimes switching from milk to BSA for blocking and diluting the primary will help develop the signal more but if you see nothing at all then I am skeptical this will help.

Have you been able to stain blots of these same samples with other antibodies? You mentioned that the secondary was known to be good but it is not clear if you used with other primaries on these HEK 293 lysates, or different lysates. In any case, I think a replacement is in order. Are you interested in trying a polyclonal antibody instead, such as ab82383?

https://www.abcam.com/index.html?datasheet=82383

I look forward to your reply.

Read More

Answer

Thank you for contacting us.
We recommend to run the gel in denaturing and reducing conditions for ab77104. Therefore, SDS PAGE gel should be used. Attached is our WB protocol and you could use it as a reference.
We use HEK 293 (huamn embryonic kidney cell line) as positive control. You could prepare the cell lysate according to the attached protocol or use ab7902 (HEK293 Human embryonic kidney cell line Whole Cell Lysate) as positive control.
I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Read More

Answer

Thank you for your enquiry.

I am sorry to confirm that these antibodies have not been tested in sandwich ELISA (sELISA). They have been tested in indirect ELISA only and I can recommend considering using them in an indirect ELISA rather than sELISA.

Sandwich ELISA procedures can be difficult to optimise and tested match pair antibodies should be used. Any tested matched pairs would be noted on the datasheet. This ensures the antibodies are detecting different epitopes on the target protein so they do not interfere with the other antibody binding. Therefore, I am sorry we are unable to guarantee our antibodies in sandwich ELISA unless they have been specifically tested for sandwich ELISA.

I am sorry I am unable to suggest any other antibodies suitable for sandwich ELISA for you on this occasion. However, I hope this information is helpful, Should you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products. I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 978396. To check the status of the order please contact our Customer Service team and reference this number. Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know. I wish you the best of luck with your research.  

Read More

Answer

Thank you for taking the time to complete our questionnaire and provide these details. The details you have kindly provided will provide us with vital information for our monitoring of product quality I appreciate the time you have spent in the laboratory and understand your concerns. It is regrettable the results have not been succesful. Reviewing the details, I am sorry there are no further tips to provide on this occasion to help improve the results. I can suggest you have regrettably recieved a bad vial. I apologise for the inconvenience and 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

Answer

Thank you for contacting us. Ab82590 and ab77104 are both Anti-ENPP2 mouse monoclonals, however,  ab82590 is from the clone [1F8] and ab77104 is from [5H3]. We are sorry if any confusion has been caused in this matter. We have no plans to discontinue either product. I am sorry to hear you have been experiencing problems with ab82590. This antibody has been tested using mouse and human samples and in paraffin embedded IHC. We would therefore expect your experiments to be working. If you wouldn't mind, could you please fill out the questionnaire I have attached in order to find out a few more details about the protocol used. This may allow me to suggest some things in order to improve the result, and will also allow us to monitor the problem and initiate any internal investigations needed in this case. If the antibody was purchased within 6 months and used according to the datasheet you will be covered by the Abpromise and will be entitled to a free of charge replacement or a credit note. I look forward to receiving your reply. 

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