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  1. Link

    cd20-antibody-ep459y-rat-igg2a-ab279300.pdf

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Immunology Adaptive Immunity B Cells CD
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Validated using a knockout cell lineRecombinant

Recombinant Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)

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Western blot - Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)
  • Western blot - Anti-CD20 antibody (ab279300)
  • Immunocytochemistry - Anti-CD20 antibody [EP459Y] (ab279300)
  • Flow Cytometry (Intracellular) - Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)
  • Immunoprecipitation - Anti-CD20 antibody (ab279300)
  • Immunocytochemistry - Anti-CD20 antibody [EP459Y] (ab279300)

Key features and details

  • Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
  • Rat monoclonal [EP459Y] to CD20 - Rat IgG2a
  • Suitable for: IP, Flow Cyt (Intra), WB, ICC
  • Knockout validated
  • Reacts with: Human

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Protein
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Recombinant Human S100A14 protein (His tag) (ab241524)
Knockout
Product image
Human MS4A1 (CD20) knockout Raji cell line (ab273871)
Primary
Product image
Anti-CD8 alpha antibody (ab4055)

View more associated products

Overview

  • Product name

    Anti-CD20 antibody [EP459Y] - Rat IgG2a
    See all CD20 primary antibodies
  • Description

    Rat monoclonal [EP459Y] to CD20 - Rat IgG2a
  • Host species

    Rat
  • Tested applications

    Suitable for: IP, Flow Cyt (Intra), WB, ICCmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

  • Positive control

    • WB: Raji and Ramos whole cell lysate and Wild-type Raji cell lysate ICC: Ramos cells. IP: Ramos whole cell lysate. Flow Cyt (intra): Ramos cells.
  • General notes

    This rat antibody has been engineered from a RabMAb parent antibody (ab78237). By necessity, some rabbit sequence is retained as part of the variable domain. When multiplexing with other rabbit-derived antibodies, using cross absorbed Fc-reactive secondary antibodies are recommended.

Properties

  • Form

    Liquid
  • Storage instructions

    Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.
  • Storage buffer

    pH: 7.2
    Preservative: 0.01% Sodium azide
    Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
  • Concentration information loading...
  • Purity

    Protein A purified
  • Clonality

    Monoclonal
  • Clone number

    EP459Y
  • Isotype

    IgG2a
  • Research areas

    • Immunology
    • Adaptive Immunity
    • B Cells
    • CD
    • Stem Cells
    • Hematopoietic Progenitors
    • Hematopoietic Stem Cells
    • Human Lineage Negative
    • Cancer
    • Tumor immunology
    • Tumor-associated antigens
    • Neuroscience
    • Processes

Associated products

  • Compatible Secondaries

    • Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed (ab150165)
    • Goat Anti-Rat IgG H&L (HRP) (ab205720)
  • Isotype control

    • Rat IgG2a, kappa monoclonal [RTK2758] - Isotype Control (ab18450)

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab279300 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
IP
1/30.
Flow Cyt (Intra)
Use a concentration of 0.2 µg/ml.
WB
1/1000.
ICC
Use a concentration of 0.2 - 1 µg/ml.
Notes
IP
1/30.
Flow Cyt (Intra)
Use a concentration of 0.2 µg/ml.
WB
1/1000.
ICC
Use a concentration of 0.2 - 1 µg/ml.

Target

  • Function

    This protein may be involved in the regulation of B-cell activation and proliferation.
  • Tissue specificity

    Expressed on B-cells.
  • Involvement in disease

    Defects in MS4A1 are the cause of immunodeficiency common variable type 5 (CVID5) [MIM:613495]; also called antibody deficiency due to CD20 defect. CVID5 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 MS4A family.
  • Post-translational
    modifications

    Phosphorylated. Might be functionally regulated by protein kinase(s).
  • Cellular localization

    Membrane.
  • Target information above from: UniProt accession P11836 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Database links

    • Entrez Gene: 931 Human
    • Omim: 112210 Human
    • SwissProt: P11836 Human
    • Unigene: 712553 Human
    • Alternative names

      • APY antibody
      • ATOPY antibody
      • B lymphocyte antigen CD20 antibody
      • B Lymphocyte Cell Surface Antigen B1 antibody
      • B-lymphocyte antigen CD20 antibody
      • B-lymphocyte cell-surface antigen B1 antibody
      • B-lymphocyte surface antigen B1 antibody
      • B1 antibody
      • Bp 35 antibody
      • Bp35 antibody
      • CD 20 antibody
      • CD20 antibody
      • CD20 antigen antibody
      • CD20 receptor antibody
      • CD20_HUMAN antibody
      • CVID 5 antibody
      • CVID5 antibody
      • Fc epsilon receptor I beta chain antibody
      • Fc Fragment of IgE high affinity I receptor for beta polypeptide antibody
      • FCER1B antibody
      • High affinity immunoglobulin epsilon receptor subunit beta antibody
      • IgE Fc receptor subunit beta antibody
      • IGEL antibody
      • IGER antibody
      • IGHER antibody
      • LEU 16 antibody
      • Leu-16 antibody
      • LEU16 antibody
      • leukocyte surface antigen Leu 16 antibody
      • Leukocyte surface antigen Leu-16 antibody
      • Ly44 antibody
      • Membrane spanning 4 domains A1 antibody
      • Membrane spanning 4 domains subfamily A member 2 antibody
      • membrane-spanning 4-domains A1 antibody
      • Membrane-spanning 4-domains subfamily A member 1 antibody
      • MGC3969 antibody
      • MS4A1 antibody
      • MS4A2 antibody
      • S7 antibody
      see all

    Images

    • Western blot - Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)
      Western blot - Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)
      All lanes : Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300) at 1/1000 dilution

      Lane 1 : Wild-type Raji cell lysate
      Lane 2 : MS4A1 knockout Raji cell lysate
      Lane 3 : A549 cell lysate
      Lane 4 : HeLa cell lysate

      Lysates/proteins at 20 µg per lane.

      Performed under reducing conditions.

      Observed band size: 33 kDa why is the actual band size different from the predicted?



      False colour image of Western blot: Anti-CD20 antibody [EP459Y] - Rat IgG2a staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab279300 was shown to bind specifically to CD20. A band was observed at 33 kDa in wild-type Raji cell lysates with no signal observed at this size in MS4A1 knockout cell line ab273871 (knockout cell lysate ab263259). To generate this image, wild-type and MS4A1 knockout Raji cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rat IgG H&L (IRDye® 800CW) preabsorbed (ab253031) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

    • Western blot - Anti-CD20 antibody (ab279300)
      Western blot - Anti-CD20 antibody (ab279300)
      All lanes : Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300) at 1/1000 dilution

      Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate
      Lane 2 : Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate

      Lysates/proteins at 20 µg per lane.

      Secondary
      All lanes : Goat Anti-Rat IgG H&L (HRP) (ab205720) at 1/5000 dilution


      Blocking/Dilution buffer: 5% NFDM/TBST.

    • Immunocytochemistry - Anti-CD20 antibody [EP459Y] (ab279300)
      Immunocytochemistry - Anti-CD20 antibody [EP459Y] (ab279300)

      Immunofluorescence staining of CD20 using ab279300 in Ramos (human Burkitt's lymphoma cell line) cells.

      The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab279300 at 0.2 µg/ml. Cells were then incubated with ab150165, Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and nuclear DNA was labelled with DAPI (shown in blue).

      The secondary only control (bottom row) was not incubated with ab279300 but otherwise processed the same.

      Images were acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

    • Flow Cytometry (Intracellular) - Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)
      Flow Cytometry (Intracellular) - Anti-CD20 antibody [EP459Y] - Rat IgG2a (ab279300)

      Flow cytometry overlay histogram showing Ramos (human Burkitt's lymphoma cell line) positive cells (left panel) and negative HEK293T (human epithelial cell line from embryonic kidney transformed with large T antigen) cells (right panel) stained with ab279300 (red line).

      The cells were fixed with 4% formaldehyde (10 min) and then permeabilized with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab279300) (1x106 in 100µl at 0.2 µg/ml) for 30 min at 22°C.

      The secondary antibody Goat anti-rat IgG H&L (Alexa Fluor® 488, pre-adsorbed) (ab150165) was used at 1/2000 for 30 min at 22°C.

      Isotype control antibody (black line) was Rat IgG2a kappa (ab18450) used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

      Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

      This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min) / permeabilized with 0.1% PBS-Triton X-100 for 15 min used under the same conditions.

    • Immunoprecipitation - Anti-CD20 antibody (ab279300)
      Immunoprecipitation - Anti-CD20 antibody (ab279300)

      CD20 was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate 10 µg with ab279300 at 1/30 dilution (2µg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab279300 at 1/1000 dilution. Goat Anti-Rat IgG (H+L), HRP) (ab205720) was used at 1/5000 dilution.

      Lane 1: Ramos whole cell lysate  10µg.

      Lane 2: ab279300 IP in Ramos whole cell lysate.

      Lane 3: Rat monoclonal IgG2a (ab18450) instead of ab279300 in Ramos whole cell lysate.

      Blocking and dilution buffer and concentration: 5% NFDM/TBST.

      Exposure time: 1 second.

    • Immunocytochemistry - Anti-CD20 antibody [EP459Y] (ab279300)
      Immunocytochemistry - Anti-CD20 antibody [EP459Y] (ab279300)

      Immunofluorescence staining of CD20 using ab279300 in Ramos (human Burkitt's lymphoma cell line) cells.

      The cells were fixed with 4% formaldehyde (10 min), permeabilized with 0.1% Triton X-100 for 5 mins and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated overnight at +4°C with ab279300 at 0.2 µg/ml. Cells were then incubated with ab150165, Goat Anti-Rat IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (shown in green) and nuclear DNA was labelled with DAPI (shown in blue).

      The secondary only control (bottom row) was not incubated with ab279300 but otherwise processed the same.

      Images were acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

    Protocols

    To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.

    Click here to view the general protocols

    Datasheets and documents

    • Datasheet download

      Download

    References (0)

    Publishing research using ab279300? Please let us know so that we can cite the reference in this datasheet.

    ab279300 has not yet been referenced specifically in any publications.

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