Overview

  • Product name
    Anti-CD81 antibody [M38] (Phycoerythrin)
    See all CD81 primary antibodies
  • Description
    Mouse monoclonal [M38] to CD81 (Phycoerythrin)
  • Host species
    Mouse
  • Conjugation
    Phycoerythrin. Ex: 488nm, Em: 575nm
  • Tested applications
    Suitable for: WB, IP, ICC, IHC-P, Functional Studies, Flow Cytmore details
  • Species reactivity
    Reacts with: Rabbit, Cat, Human
  • Immunogen

    Tissue, cells or virus corresponding to Human CD81. MOLT4 cell line

  • General notes
    The reagent is designed for Flow Cytometry analysis of human blood cells using 20 µl reagent / 100 µl of whole blood or 106 cells in a suspension. The content of a vial (2 ml) is sufficient for 100 tests.


    In human MOLT-4 T-cell line the ab81436 inhibits syncytium formation induced by coculture with human T-cell leukemia virus type 1 (HTLV-1)-positive human T-cell lines.

Properties

Applications

Our Abpromise guarantee covers the use of ab81436 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 Use at an assay dependent concentration. Predicted molecular weight: 26 kDa.
IP Use at an assay dependent concentration.
ICC Use at an assay dependent concentration.
IHC-P Use at an assay dependent concentration.
Functional Studies Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

The reagent is designed for Flow Cytometry analysis of human blood cells using 20 ul reagent / 100 ul of whole blood or 106 cells in a suspension.

 

 

 

ab91357 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

 

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

References

This product has been referenced in:
  • Escola JM  et al. Selective enrichment of tetraspan proteins on the internal vesicles of multivesicular endosomes and on exosomes secreted by human B-lymphocytes. J Biol Chem 273:20121-7 (1998). Read more (PubMed: 9685355) »
  • Imai T & Yoshie O C33 antigen and M38 antigen recognized by monoclonal antibodies inhibitory to syncytium formation by human T cell leukemia virus type 1 are both members of the transmembrane 4 superfamily and associate with each other and with CD4 or CD8 in T cells. J Immunol 151:6470-81 (1993). IP ; Human . Read more (PubMed: 8245480) »
See all 3 Publications for this product

Customer reviews and Q&As

1-5 of 5 Abreviews or Q&A

Answer

Thank you for your reply. I appreciate the time taken to submit protocol information and I would like to make some suggestions to the protocol in an attempt to improve the results obtained. -I would recommend fixation for shorter time. Fixing in paraformaldehyde for more than 10-15 min will cross link the proteins to the point where antigen retrieval may be required to ensure the antibody has free access to bind and detect the protein. As no Antigen retrieval is used, I would then recommend fixation for 10 minutes instead of 30. After fixation, washing is required, preferably with PBS / PBS with 1% BSA. -As the target the antibody detects is a membrane protein, permeabilization is not needed. This step is only required when the antibody needs access to the inside of the cells to detect the protein. By permeabilizing the membrane may be destroyed, making the antibody unable to reach the target. I would suggest repeating the staining without permeabilization agent. -The antibody amount should be enough to cover the cells, and there isn’t a recommended dilution, as it has to be optimised for each case. I would then try performing the staining changing the fixation step and avoiding permeabilization, using several antibody dilutions, like the ones already used, checking which one gives the best results. You will find below the link to our ICC protocol from our website, which can be useful: https://www.abcam.com/index.html?pageconfig=resource&rid=11417 -We also recommend incubating the antibody over night at 4C to get a better staining. I would also appreciate if you could send me an image as well as the order details (order number, batch number) in order to better understand why this antibody did not perform as stated in the datasheet. I hope this information is useful. Please do not hesitate to contact us if you need further advice or information.

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching 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.

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Answer

Thank you for contacting us. If you are not able to easily pellet the exosomes, I would indeed recommend to add 20 µl of the antibody solution to the exosomes in 100 µl PBS. You may need to increase the incubation time and or the volume of ab81436 added, in order to optimise the staining conditions for this particular setting. To our knowledge, this has never been done before, so I am sorry that we cannot give you more detailed information. However, I trust that you will succeed! Unfortunately, it is NOT possible to determine the EXACT concentration of ab81436: The concentration of the specific TAPA1 antibodies has not been determined before conjugation. Even if it had been, this concentration would be different from the concentration of the antibody after the conjugation procedure, as there is always a loss during the purification steps. Determining the protein concentration after the conjugation does not result in accurate measurements, because Phycoerythrin (PE) is a huge protein itself and thus would give false results, as my colleagues tried to explain earlier (in case you did not receive these emails, see below). We would therefore suggest to use the PE isotype control ab81200 in the same dilution than ab81436. For example, if you add 20 µl of ab81436 to 100 µl of your sample, than it is recommended to also use 20 µl of the isotype control ab81200 for 100 µl of sample. Also, I would like to draw your attention to the criteria to record information about the experimental overview and samples (including controls) set out by the flow cytometry community (e.g. http://www.flowcytometryuk.org/). These criteria are known as the Minimum Information about a Flow Cytometry Experiment (MIFlowCyt), and contain valuable information about isotype controls and FMO controls. I hope this information is helpful to you. Please do not hesitate to contact me if you need any further advice or information in this regard. Previously: I can confirm that the concentration of this antibody was not determined. Regrettably, it is not possible to correctly measure the antibody concentration of products with antibodies conjugated to proteins (such as PE). /If there are concentrations given for conjugated antibodies, it is usually the IgG concentration measured before the conjugation took place and therefore only an estimate./ Unfortunately, we do not currently have more information about the concentration of ab81436./ Antibody concentration is usually determined by measuring the protein concentration. This is not useful in this case since PE is also a protein.

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Answer

Thank you for contacting us. I can confirm that the PE-conjugated CD81 antibody ab81436 is designed for Flow Cytometry analysis of human blood cells, and if the volume of your whole blood sample is 100 µl as indicated, we would suggest the following procedure: centrifuge the 100 µl of whole blood, remove the supernatant and keep the pellet optional step: perform a red blood lysis (see attached for details) add 20 µl of the ready-to-use antibody incubate and wash as usual and proceed with analysis Also, if you perform you perform flow cytometric analysis, we would recommend to optimise the instrument settings in order to detect the correct particles, as exosomes can be much smaller than cells. For your questions about the isotype controls: Isotype controls are a type of control designed to measure the level of non-specific background signal caused by primary antibodies. The background signal is usually the result of immunoglobulins binding non-specifically to Fc receptors present on the cell surface. For example, antibodies raised in mice, particularly those of the IgG2a isotype, will bind strongly to some human leukocytes regardless of the test antibody specificity. In this case you would need a mouse IgG2a isotype control for use with human cells or tissues. Thus, the isotype control antibody should always match the primary antibody’s host species, isotype, AND conjugation. It is therefore not advisable to use a FITC-conjugated IgG1 isotype control for a PE-conjugated antibody. Usually, if the concentration of the specific antibody is unknown, it can be recommended to use the isotype control in excess, i.e. to use a lower dilution for the isotype control than for the antibody of interest. Although isotype controls are frequently used, it is not the only type of control to determine non-specific binding and positivity for the marker of interest. This control is termed fluorescence minus one or FMO and works like an isotype control without the actual antibody. FMO controls are set up by leaving out one of the antibodies in your staining panel. This control might be also an option for your experiments and you may find more information about this type of control in the latest publications or on flow cytometry websites, such as http://www.isac-net.org or http://www.cyto.purdue.edu/ . I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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Answer

Thank you for your inquiry. Unfortunately, we do not have information about the concentration of ab81436. Antibody concentration is usually determined by measuring the protein concentration. This is not useful in this case since PE is also a protein. This antibody is tested and optimized for usage as stated on the datasheet: 20ul per 10 million cells and 100 tests are in one unit. Regarding the isotype control, I conducted a search through our website for a compatible one for ab81436 and found product ab81200. Please have a look at its datasheet: https://www.abcam.com/Mouse-IgG1-PE-Isotype-control-ab81200.html I hope this information is helpful and wish you good luck with your research. Please do not hesitate to contact me with any further questions.

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