Recombinant
RabMAb

Recombinant Anti-CD81 antibody [EPR4244] (ab109201)

Rabbit recombinant monoclonal CD81 antibody [EPR4244]. Validated in WB and tested in Mouse, Rat, Human. Cited in 7 publication(s). Independently reviewed in 4 review(s).

Overview

  • Product name
    Anti-CD81 antibody [EPR4244]
    See all CD81 primary antibodies
  • Description
    Rabbit monoclonal [EPR4244] to CD81
  • Host species
    Rabbit
  • Tested applications
    Suitable for: WBmore details
    Unsuitable for: Flow Cyt,ICC/IF,IHC-P or IP
  • Species reactivity
    Reacts with: Mouse, Rat, Human
  • Immunogen

    Synthetic peptide within Human CD81 aa 150-250. The exact sequence is proprietary.
    Database link: P60033

  • Positive control
    • WB: Ramos, JAR, U87-MG, IM-9, peripheral blood mononuclear cell (PBMC), C6, PC-12, and RAW264.7 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

Applications

Our Abpromise guarantee covers the use of ab109201 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: 26 kDa.

For unpurified use at 1/500 - 1/1000.

  • Application notes
    Is unsuitable for Flow Cyt,ICC/IF,IHC-P or IP.
  • Target

    • Function
      May play an important role in the regulation of lymphoma cell growth. Interacts with a 16-kDa Leu-13 protein to form a complex possibly involved in signal transduction. May acts a the viral receptor for HCV.
    • Tissue specificity
      Hematolymphoid, neuroectodermal and mesenchymal tumor cell lines.
    • Involvement in disease
      Defects in CD81 are the cause of immunodeficiency common variable type 6 (CVID6) [MIM:613496]; also called antibody deficiency due to CD81 defect. CVID6 is a primary immunodeficiency characterized by antibody deficiency, hypogammaglobulinemia, recurrent bacterial infections and an inability to mount an antibody response to antigen. The defect results from a failure of B-cell differentiation and impaired secretion of immunoglobulins; the numbers of circulating B cells is usually in the normal range, but can be low.
    • Sequence similarities
      Belongs to the tetraspanin (TM4SF) family.
    • Post-translational
      modifications
      Not glycosylated.
    • Cellular localization
      Membrane.
    • Information by UniProt
    • Database links
    • Alternative names
      • 26 kDa cell surface protein TAPA 1 antibody
      • 26 kDa cell surface protein TAPA-1 antibody
      • 26 kDa cell surface protein TAPA1 antibody
      • CD 81 antibody
      • CD81 antibody
      • CD81 antigen (target of antiproliferative antibody 1) antibody
      • CD81 antigen antibody
      • CD81 molecule antibody
      • CD81_HUMAN antibody
      • CVID6 antibody
      • S5.7 antibody
      • TAPA 1 antibody
      • TAPA1 antibody
      • Target of the antiproliferative antibody 1 antibody
      • Tetraspanin 28 antibody
      • Tetraspanin-28 antibody
      • Tetraspanin28 antibody
      • Tspan 28 antibody
      • Tspan-28 antibody
      • Tspan28 antibody
      see all

    Images

    • All lanes : Anti-CD81 antibody [EPR4244] (ab109201) at 1/700 dilution (unpurified)

      Lane 1 : Ramos cell lysate
      Lane 2 : U87-MG cell lysate
      Lane 3 : PC-12 cell lysate
      Lane 4 : Raw264.7 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 26 kDa
      Observed band size: 20 kDa
      why is the actual band size different from the predicted?

    • All lanes : Anti-CD81 antibody [EPR4244] (ab109201) at 1/2000 dilution (purified)

      Lane 1 : Ramos cell lysate
      Lane 2 : U87-MG cell lysate
      Lane 3 : PC-12 cell lysate
      Lane 4 : Raw264.7 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

      Predicted band size: 26 kDa
      Observed band size: 20 kDa why is the actual band size different from the predicted?

    • All lanes : Anti-CD81 antibody [EPR4244] (ab109201) at 1/1000 dilution (unpurified)

      Lane 1 : Ramos cell lysate
      Lane 2 : JAR cell lysate
      Lane 3 : U87-MG cell lysate
      Lane 4 : IM-9 cell lysate
      Lane 5 : Peripheral blood mononuclear cell (PBMC) cell lysate
      Lane 6 : C6 cell lysate
      Lane 7 : PC-12 cell lysate
      Lane 8 : RAW264.7 cell lysate

      Lysates/proteins at 10 µg per lane.

      Secondary
      All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721)

      Predicted band size: 26 kDa

    References

    This product has been referenced in:
    • Im EJ  et al. Sulfisoxazole inhibits the secretion of small extracellular vesicles by targeting the endothelin receptor A. Nat Commun 10:1387 (2019). Read more (PubMed: 30918259) »
    • Yu J  et al. Detection of Exosomal PD-L1 RNA in Saliva of Patients With Periodontitis. Front Genet 10:202 (2019). Read more (PubMed: 30923536) »
    See all 9 Publications for this product

    Customer reviews and Q&As

    1-10 of 11 Q&A

    Question


    The images that I send to you are T cells which express tones of CD81 that is why I didn't send any picture of HeLa cells (though based on our experience one can detect CD81 in HeLa cells).
    The problem that I am having with the rabbit Ab is that he doesn't correspond to CD81 staining in T cells. CD81 is mostly express at the pm in those cells and I have been working to understand CD81 down regulation in HIV-1 infected cells.
    IF staining protocol (standard condition):
    Briefly, CEMss cells (T cells) have been allowed to stick on poly-L-lysine treated MatTek dishes for 2h then wash once with PBS, fix using PFA 4% (wash then) for 10 min at 37 deg C, permeabilized (PBS + 1% BSA + 0.2% Triton) for 20 min at 37 deg C, primary Ab were incubated 1h at RT (rabbit anti-CD81 diluted 100 times, while mouse anti-CD81 diluted 500 times), wash , incubated with secondary Ab at RT for 30 min, wash, 2 ml PBS. Then I analyzed my cells on a deconvolution microscope.
    Western Blot (standard condition):
    When I used the Ab for WB application, I used CEMss lysates (T cells line lysed in PBS-1% Brij 96/97 containing PI) (infected or not). Using your Ab I couldn't see anything but when reprobing using our anti-CD81 I would be able to see the bands … (Conditions: 10% SDS-acrylamide gel, migration 120 V (mini-Protean system), semi-dry transfer (25V for 1h30) onto PVDF membrane, wash once TBS-T, Block in 5% milk in TBS-T, primary ON at 4 deg C, was 3 times, secondary HRP for 30 min at RT, wash 3 times, ECL.
    I also was doing IP of CD81 and I couldn't see anything either (I was using the rabbit Ab at a dilution of 1/1000). I was IPing using the ms anti-CD81 and try to detect with the Rabbit again nothing clear in my hands.
    Let me know if you have more questions/concerns based on my protocol.
    Thank you for your reply.
    Best regards,

    Read More
    Answer

    Thank you for your reply and protocol information.
    If we cannot get this antibody to work as stated on the datasheet we will replace it with a new vial of the same antibody or a different antibody of your choosing free of charge as indicated in our AbPromise guarantee. Given that, I have some more questions and protocol tips.
    Are you reducing and denaturing your sample prior to Western blot? We recommend boiling for 5 minutes at 95oC in beta-mercaptoethanol or DTT + SDS prior to loading. Since there is a disulfide bond within the residues that are recognized by this antibody, if that disulfide bond is not broken via reduction/denaturation the antibody may not properly recognize its epitope.
    For IF: In our experience testing our antibodies, Triton-X-100 is not appropriate for the use of membrane-associated antigens (such as CD81) since it destroys membranes. Since the specific epitope for ab109201 is extracellular, you should not have to permeabilize the cell membrane in order for the antibody to detect its antigen.
    For IP: As stated on the datasheet, this antibody is unsuitable for flow cytometry, IHC-P, and IP, explaining why it did not work in IP for you. The unsuitability of this antibody in these applications where CD81 is in a native conformation is most likely due to the fact that the antibody was raised against a synthetic peptide corresponding to an extracellular domain of CD81 that contains a disulfide bond (forming a loop) that is harder for the antibody to recognize in native 3D form compared to a linearized (ie. reduced, denatured for WB) form.
    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 contacting Abcam.
    I am sorry to hear that this antibody is not working for you. As this was a free antibody given through our RabMab promotion, we cannot issue a refund in this case. However, if we cannot work with you to get ab109201 to work as indicated on the datasheet we will happily send you a free of charge replacement of the same antibody or another of your choosing.
    Thank you kindly for including the ppt file with your IF results. My main concern is with the lack of a band in the WB, since there is definitely a positive signal in your IF, just not colocalized with the other CD81 antibody you used. Since these two antibodies were raised against different immunogenic sequences for the same CD81 protein, it may be that some cells express CD81 in a form more recognizable by one antibody over the other.
    Thus, in order to help troubleshoot these problems, could you provide details regarding your sample, protocol and troubleshooting steps that have been taken, specifically for WB. These will not only help me to fully understand the problem but are essential should further testing of this product be needed as well. Could you also provide the Abcam order confirmation number or the PO and date of purchase for this product?
    Based on the information you have provided, I have a few recommendations. According to the Human Protein Atlas database for CD81 (http://www.proteinatlas.org/ENSG00000110651/cell), HeLa cells have very weak, if any, expression of CD81. The positive control cell lines that we have tested are Ramos, JAR, U87 MG, IM 9, Peripheral blood mononuclear cell (PBMC), C6, PC 12, and RAW264.7 cell lysates. I highly recommend trying one of these positive controls. I look forward to your reply. Please do not hesitate to contact us if you need any more advice or information.
    Below is a link with further troubleshooting steps if you have not performed them already:
    https://www.abcam.com/index.html?pageconfig=resource&rid=11352
    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

    Question

    Catalog number(s): 109201
    Product(s): Rabbit monoclonal [EPR4244] to TAPA1

    Survey results
    ----------------------------------------------------------------------------------
    Telephone section
    Q. Did you contact Abcam's Scientific Support by telephone in reference to this complaint?
    A. Yes
    Q. Was it clear from the phone options which number you needed to press to get your issue resolved?
    A. Yes

    Protocol advice section
    Q. Did Abcam's Scientific Support give you protocol advice?
    A.Yes
    Q. How did you feel about being given protocol advice?
    A.I was not expecting to be given protocol advice, but was happy to receive this
    Q. Did the product work successfully after following the advice?
    A.No
    Q. Why have you not tried the protocol advice? (Select all that apply.)
    A.

    Free of charge replacement section
    Q. Did Abcam's Scientific Support give you a free of charge replacement?
    A. Yes

    Q. Did the replacement product work successfully?
    A.Yes
    Credit note / refund section
    Q. Did Abcam's Scientific Support give you a credit note or a refund?
    A.Yes

    Q. Would you have preferred a different outcome from receiving a credit note or refund?
    A.No
    Overall satisfaction section
    Q. How satisfied are you with how the complaint was handled?
    A.Very satisfied
    Q. Could we have done anything to better resolve the problem?
    A.
    Q. Would you mind if our scientific support team contacted you to better understand what happened and reach an agreeable solution?
    A.No, I would not mind
    Q. Please share with us any final thoughts you may have about Abcam, the complaints process, or our products.
    A.

    Read More
    Answer

    Thank you for your participation in Abcam's post complaint survey.

    Please let me know if there is anything else I can do for you.

    Also, if you have further questions to any of our products (e.g. primary antibodies, lysates, secondary antibodies, kits, controls, gels, proteins/peptides, slides, biochemicals, conjugation kits), please let me know as well.

    Read More

    Answer

    Vielen Dank für Ihre Antwort.

    Es tut mir leid, dass Sie Probleme mit diesen beiden Antikörpern hatten. Wie per Emailbesprochen, habe ich eine kostenlose Ersatzlieferung für Sie in Auftrag gegeben. Sie hat die Referenznummer xxx fuer ab7090 und ab93485, und sollte diese Woche bei Ihnen ankommen.

    Ich wünsche Ihnen viel Erfolg für Ihr Projekt. Bitte zögern Sie nicht, sich wieder bei uns zu melden, falls Sie weitere Fragen haben.

    Read More

    Answer

    Vielen Dank fuer Ihre email.

    Ich habe nach Sekundaerantikoerpern in unserem Katalog geschaut. Wir haben zahlreiche, aber die folgenden haben wir momentan im Lager:

    ab16284 donkey anti-rabbit, HRP(https://www.abcam.com/Rabbit-IgG-secondary-antibody-ab16284.html)

    ab7090: goat anti-rabbit, HRP pre-absorbed (https://www.abcam.com/Rabbit-IgG-secondary-antibody-H-L-Pre-Adsorbed-ab7090.html)

    ab6795: sheep anti-rabbit, HRP (https://www.abcam.com/Rabbit-IgG-secondary-antibody-H-L-ab6795.html)

    Alle diese Antikoerper waeren geeignet fuer den Primaerantikorper ab93485.

    Bitte sagen Sie mir doch Bescheid welchen Sekundaerantikoerper Sie bevorzugen wuerden, so dass ich die Ersatzlieferung arrangieren kann.

    Vielen Dank, und ich freue mich wieder von Ihnen zu hoeren.

    Read More

    Answer

    Vielen Dank fuer Ihre email.

    In diesem Falle koennte ich Ihnen auch einen Ersatz fuer den Primaerantikoerper anbieten.

    Wuerden Sie einen anderen C81 Antikoerper probieren wollen, da wir kein neues Batch fuer ab109201 in Lager haben? Zum Beispiel:

    ab79559 (https://www.abcam.com/TAPA1-antibody-M38-ab79559.html)
    ab93485 (https://www.abcam.com/TAPA1-antibody-ab93485.html)
    ab35026 (https://www.abcam.com/TAPA1-antibody-1D6-Azide-free-ab35026.html)
    ab128202 (https://www.abcam.com/TAPA1-antibody-ab128202.html)

    Gemaess Ihrer Wahl des Primaerantikoerpers, koennen wir dann einen neuen Sekundaerantikoerper fuer Sier heraussuchen. Sagen Sie mir doch bitte Bescheid bezueglich Ihrer Auswahl.

    Ich freue mich wieder von Ihnen zu hoeren.

    Read More

    Answer

    Vielen Dank fuer Ihre email und die extra Informationen.

    Ja, Sie haben Recht - die Bande bei 26 kDa ist nicht zu sehen. Das Protein CD81 ist ein Membranprotein mit 4 helikalen Domainen. Hat der Transfer funktioniert?

    Ich wuerde vorschlagen, bis zu max. 0.1% SDS zum Transferbuffer hinzuzugeben, da das fuer hydrophobe Proteine hilfreich sein kann und es verhindert, dass diese in der Membran schon prezipitieren.

    Ein anderer Vorschlag waere (falls Sie das nicht schon gemacht haben), den Transfer mit Ponceau S (fuer die Membran) und Coomassie Blue (fuer das Gel) zu ueberpruefen. Entweder das Gel oder die Membran sollte wenigstens eine Bande bei 26 kDa zeigen.

    Ich nehme an, dassCD81 in humanen PBMCs expressiert ist?

    Die anderen Banden, die Sie detektiert haben, scheinen vom Sekundaerantikoerper zu stammen. Diesen Antikoerper kann ich Ihnen auf jeden Fall kostenlos ersetzen.

    Sagen Sie mir doch bitte Bescheid bezueglich der Fragen, und dann koennen wir entscheiden wie wir mit dem Primaerantikoerper verfahren werden.

    Ich freue mich wieder von Ihnen zu hoeren und Ihnen bei der Loesung dieses Problems zu helfen.

    Read More

    Answer

    Thank you for getting back to me and for confirming the order details and the batch number.
    I would like to reassure you that our Abpromise applies to your complaint since you purchased this product within the guarantee period. This means that in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
    Though, you have kindly provided some details, it would be much appreciated if I could get some more information which would help me identify the source of the problem.
    Could you provide some further details of the protocol used and complete the following form (attached as a word document). It would be much appreciated if you could attach an image to the response.
    I am particularly interested in the followings:
    - sample preparation,
    - lysis buffer,
    - positive control,
    - loading control
    Thank you for your understanding and co-operation in this matter. I look forward to hearing from you and hope to solve this problem as soon as possible.

    Read More

    Answer

    Vielen Dank fürIhre Anfrage.
    Es tut mir leid, dassdiese Antikoerpern IhnenProbleme bereiten. Ich vermute, dass das Problem beim Sekundaerantikorrper liegt.

    Ich hab noch ein paar Fragen an Sie:

    1) Was fuer Proben benutzen Sie (Zell/Gewebe-type, Spezies)?
    2) Haben Sie mal probiert mit BSA zu blocken?
    3) Haben Sie auch eine noch hoehere Verduennung des Sekundaerantikoerpers versucht (z.B. 1/20,000 oder 1/50'000)?
    4) Tritt das gleiche Problem auch mit anderen Primaerantikoerpern auf?
    5) Was ist Ihre Bestellnummer?

    Die Bande um die 50-60 kDakoennte endogenes IgG (heavy chain) sein - falls Sie mit Gewebeproben arbeiten.

    Der Sekundaerantikoerper sollte eigentlich kein Signal geben wenn der Primaerantikoerper weggelassen wurde.

    Ich freue mich wieder von Ihnen zu hoeren und Ihnen bei der Loesung dieses Problems zu helfen.

    Read More

    Question

    Hi there,



    A couple of months ago I purchased your rabbit anti-CD81 (anti-TAPA-1) antibody (ab109201) which is guaranteed to detect human CD81 by western blot. I have been unable to detect CD81 in blots and wonder if I am doing something incorrectly as another one of your antibodies (ab79559) also failed to wprk in western blot.



    I am using 2 cell lines (Huh7.5 and 293T cells) and I have shown by flow cytometry that both contain surface CD81:

    - they both strongly bind another one of your anti-CD81 antibodies (ab79559 - flow cytometry and WB guaranteed);

    - they both bind HCV E2 (the receptor of which is CD81);

    - cells pre-incubated withab79559 no longer bind HCV E2 (as CD81 is blocked by your antibody).



    I wanted to confirm the presence of CD81 by WB so performed SDS-PAGE withwhole cell lysates (from Huh7.5, 293T, Raji, Jurkat and other cell lines) using standard protocols, transferred proteins onto nitrocellulose, blocked for 2 h with 10 % milk, and probed overnight withab79559 at 1:1000 dilution in 5 % milk in TBS-T, followed by anti-mouse-HRP. I even got no signal, even when reprobed at 1:100 dilution (incidentally, the problem is not with the secondary as that works with other antibodies).



    I assumed thatab79559 must detect a 'correctly folded' epitope as it works with flow cytometry and that this must be altered by SDS-PAGE so I tried your WB-specific anti-CD81 antibody (ab109201). WB was performed as above, but using an anti-rabbit-HRP secondary, and again I got no signal.





    I then transiently transfected 293T, 293T/17 and Huh7.5 cells with myc-DDK-tagged CD81 on the CMV promoter (Origene RC217508), and after 48 h expression performed SDS-PAGE and WB using whole cell lysates of transfected and non-transfected cells. I then probed one blot with your WB-guaranteed anti-CD81 antibody (ab109201) and another blot with an anti-flag antibody. The anti-flag antibody strongly detected CD81 in lanes from CD81-transfected cells, but nothing in non-transfected lanes. Strangely, your anti-CD81 antibody detected nothing - even the transfected lanes had no signal.



    I had begun to think that CD81 perhaps did not run into the gel during SDS-PAGE (as membrane proteins can sometimes be temperamental), but I am perplexed that I can detect tagged CD81 with the anti-tag antibody, but not with your anti-CD81. Please can you tell me if there is something that I have failed to do correctly for the detection of CD81 by WB so that I can test the antibody again.



    Kind regards,

    Read More
    Answer

    Thank you for your enquiry regarding ab109201 and for taking the time to provide some useful details of the experiments. I am very sorry to hear that you are having problems with this antibody.
    As our Abpromise indicates, in the event that a product is not functioning in the applications/species cited on the product data sheet (and the problem has been reported within 6 months of purchase) we will happily offer a credit note/refund to the value of the product purchased.
    Could you please provide the batch and order numbers for ab109201?
    I look forward to hearing from you and hope to solve this problem as soon as possible.

    Read More

    1-10 of 11 Q&A

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