Overview

  • Product name
    Anti-CD39 antibody [EPR3678(2)]
    See all CD39 primary antibodies
  • Description
    Rabbit monoclonal [EPR3678(2)] to CD39
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WBmore details
    Unsuitable for: Flow Cyt,ICC,IHC-P or IP
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human CD39 aa 350-450. The exact sequence is proprietary.

  • Positive control
    • WB: Human placenta, rat hippocampus, rat pancreas, mouse placenta and mouse urinary bladder tissue lysates; IM-9 and HUVEC cell lysates.
  • 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

Associated products

Applications

Our Abpromise guarantee covers the use of ab108248 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: 58 kDa.Can be blocked with Human CD39 peptide (ab228815).
  • Application notes
    Is unsuitable for Flow Cyt,ICC,IHC-P or IP.
  • Target

    • Function
      In the nervous system, could hydrolyze ATP and other nucleotides to regulate purinergic neurotransmission. Could also be implicated in the prevention of platelet aggregation. Hydrolyzes ATP and ADP equally well.
    • Tissue specificity
      Expressed primarily on activated lymphoid cells. Also expressed in endothelial tissues. The vascular isoform and the placental isoform II are present in both placenta and umbilical vein, whereas placental isoform I is present in placenta only.
    • Sequence similarities
      Belongs to the GDA1/CD39 NTPase family.
    • Post-translational
      modifications
      The N-terminus is blocked.
      Palmitoylated in the N-terminal part.
    • Cellular localization
      Membrane.
    • Information by UniProt
    • Database links
    • Form
      There are 3 isoforms produced by alternative splicing.
    • Alternative names
      • ATPDase antibody
      • CD 39 antibody
      • CD39 antibody
      • CD39 antigen antibody
      • DKFZp686D194 antibody
      • DKFZp686I093 antibody
      • Ecto apyrase antibody
      • Ecto ATP diphosphohydrolase antibody
      • Ecto-apyrase antibody
      • Ecto-ATP diphosphohydrolase 1 antibody
      • Ecto-ATPase 1 antibody
      • Ecto-ATPDase 1 antibody
      • Ectonucleoside triphosphate diphosphohydrolase 1 antibody
      • ENTP1_HUMAN antibody
      • ENTPD 1 antibody
      • ENTPD1 antibody
      • FLJ40921 antibody
      • FLJ40959 antibody
      • Lymphoid cell activation antigen antibody
      • NTPDase 1 antibody
      • NTPDase1 antibody
      • SPG64 antibody
      see all

    Images

    • All lanes : Anti-CD39 antibody [EPR3678(2)] (ab108248) at 1/1000 dilution (purified)

      Lane 1 : Rat hippocampus
      Lane 2 : Rat pancreas

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size: 58 kDa
      Observed band size: 70,74 kDa
      why is the actual band size different from the predicted?



      Blocking and diluting buffer and concentration 5% NFDM/TBST.

    • All lanes : Anti-CD39 antibody [EPR3678(2)] (ab108248) at 1/1000 dilution (purified)

      Lane 1 : Mouse placenta
      Lane 2 : Mouse urinary bladder

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size: 58 kDa
      Observed band size: 70 kDa why is the actual band size different from the predicted?


      Exposure time: 15 seconds


      Blocking and diluting buffer and concentration 5% NFDM/TBST.

      The PKC beta 2 recombinant protein is GST-tagged, so the molecular weight is higher.

    • Lane 1 : Anti-CD39 antibody [EPR3678(2)] (ab108248) at 1/100 dilution (purified)
      Lane 2 : Anti-CD39 antibody [EPR3678(2)] (ab108248) at 1/1000 dilution (purified)

      All lanes : HUVEC whole cell lysate

      Lysates/proteins at 20 µg per lane.

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

      Predicted band size: 58 kDa
      Observed band size: 70,74 kDa why is the actual band size different from the predicted?


      Exposure time: 3 minutes


      Blocking and diluting buffer and concentration 5% NFDM/TBST.

      Ref: PMID: 18693757

    • All lanes : Anti-CD39 antibody [EPR3678(2)] (ab108248) at 1/1000 dilution (unpurified)

      Lane 1 : Human placenta lysate
      Lane 2 : IM-9 cell lysate

      Predicted band size: 58 kDa

    References

    This product has been referenced in:
    • Kong LR  et al. Decrease of Perivascular Adipose Tissue Browning Is Associated With Vascular Dysfunction in Spontaneous Hypertensive Rats During Aging. Front Physiol 9:400 (2018). Read more (PubMed: 29720945) »
    • Villa-Bellosta R  et al. Novel phosphate-activated macrophages prevent ectopic calcification by increasing extracellular ATP and pyrophosphate. PLoS One 12:e0174998 (2017). Read more (PubMed: 28362852) »
    See all 6 Publications for this product

    Customer reviews and Q&As

    1-2 of 2 Abreviews or Q&A

    Question

    Dear abcam members,



    Follow attached the WB Questionnaire, 2 images and some details regarding the western Blotting.



    Hope you can help us as soon as possible,



    Best regards,



    Ana Andrade

    WB Questionnaire



    1) Abcam product code: ab108248



    2) Abcam order reference number or product batch number GR60408-3



    3) Description of the problem – Unspecific bands



    4) Sample preparation:

    Type of sample (whole cell lysates, fraction, recombinant protein…): whole cell lysate (mesenchymal stromal cells)

    Lysis buffer: RIPA buffer

    Protease inhibitors: Complete protease inhibitors cocktail (Roche)

    Phosphatase inhibitors: no

    Reducing agent: Mercaptoethanol

    Boiling for ≥5 min? YES

    Protein loaded: 6x105 cells/ lane or 3x105/ lane

    Positive control - MSCs are positive as we already observed by FACS and immunofluorescence but there are many bands. Caco-2 was added as another positive control, but we couldn´t make any conclusions.

    Negative control - I didn´t use negative control



    5) Percentage of gel – 7,5%

    Type of membrane: PVDF

    Protein transfer verified: YES

    Blocking agent and concentration 5% of milk in PBS + 0.3%Tween 20

    Blocking time : incubated overnight in the cold room while shaking

    Blocking temperature: 4ºC



    6) Primary antibody (If more than one was used, describe in “additional notes”): Rabbit monoclonal antibody to CD39 (IgG1)

    Concentration or dilution 1:1000

    Diluent buffer: 5% of milk in PBS + 0.3%Tween 20

    Incubation time 60 minutes while shaking

    Incubation temperature: RT





    7) Secondary antibody:

    Species: Goat

    Reacts against: rabbit IgG

    Concentration or dilution: 1:5000

    Diluent buffer: 5% of milk in PBS + 0.3%Tween 20

    Incubation time: 60 minutes

    Incubation temperature: RT

    Fluorochrome or enzyme conjugate: horseradish peroxidase



    8) Washing after primary and secondary antibodies:

    Buffer: PBS + 0.3%Tween 20

    Number of washes: 2 times and then 3 times for 10 minutes with PBS + 0.3%Tween 20 at RT while shaking



    9) Detection method

    10) How many times have you run this staining? 1

    Do you obtain the same results every time?

    What steps have you altered to try and optimize the use of this antibody? None yet.



    Two images follow attached (the first was exposed for 1 minute and the second for 12 mins)

    The Caco-2 (colorectal carcinoma cell line is supposed to be positive also).

    We checked the MSCs with immunofluorescence before doing the Western Blotting to be sure that they are positive, but we can not conclude anything from the bands we got.

    Read More
    Answer

    Thank you for taking time to complete our questionnaire. I am sorry to hear that this antibody is not providing satisfactory results.

    The details provided will enable us to investigate this case and will provide us with vital information for monitoring product quality.

    Having reviewed this case, I would like to offer some suggestions to help optimise the results from ab108248 Anti-CD39 antibody [EPR3678(2)]. I would also appreciate if you can confirm some further details:


    1.As this is a multipass membrane protein, which tent to aggregate in the loading buffer when boiled at high temperature due to their hydrophobic nature, I would like to recommend lowering the boiling temperature down to 60C and prolonging the boiling time up to 10 min. In addition, use DTT instead of beta-mercaptoethanol as this kind of aggregation is favoured by b- ME.


    2. As this antibody is quite sensitive, and as you are working with presumably positive cells, please measure the protein concentration. In order to avoid over-loading and therefore to decrease the side bands, I would like to suggest loading 20 to 30 ug protein per lane.



    3. In order to prevent a masking of your target, please do not block longer than one hour. Overnight blocking helps to keep down a high background but here it is about obtaining a signal. Do not over-block. Long blocking incubations, particularly with nonfat dry milk at 2% or higher, can cause loss of target protein from the membrane (J. Immunol. Meth. 122:129-135, 1989). Should the suggestions not improve the results, please do let me know.



    4.To increase the antibodies sensitivity and specificity, I would like to suggest to incubate overnight with a higher dilution of the antibody at 1/5000.


    5. According to our datasheet, this antibody is unsuitable for Flow Cyt,ICC,IHC or IP. However, I couldn't find any evidence regarding his statement. Therefore, your results in FACS or ICC may be right. I will look into this by contacted the lab.


    6. Are you running the gel under reducing conditions? Most of our antibodies are tested and optimised to work under reducing conditions, and we usually state on our datasheet if the gel should be run under non-reducing conditions. I would therefore suggest to run a SDS-PAGE under reducing conditions. Otherwise it may be that the antibody may not be able to detect its target.


    7. Can you rule out that the secondary antibody is causing the problem and reacting with your samples?


    8. Last but not least:For a better saturation of the membrane, I would recommend a blocking buffer containing 3% BSA for 2 hours at room temperature. BSA can sometimes give better results than milk.Do not add Tween or another detergent as this will weaken the desired blocking effect.


    Should the suggestions not improve the results, please do let me know.


    In the event that a product is not functioning in the species and applications cited on the product datasheet (and the problem has been reported within 6 months of purchase), we would be pleased to provide a free of charge replacement, credit note, or refund.

    I hope this information is helpful, and I thank you for your cooperation.

    Read More

    Question
    Answer

    Thank you for calling us and for alerting us to the problem you are experiencing with our product. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

    As we discussed on the phone, I am our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

    I look forward to receiving your reply.

    Read More

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