Anti-CD20 antibody [EP459Y] ab78237 is a rabbit monoclonal antibody that is used in CD20 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone EP459Y has been tried and trusted by researchers since 2009 and is cited in >100 publications
- Specificity confirmed with MS4A1 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
ICC/IF | Flow Cyt (Intra) | IP | WB | IHC-P | |
---|---|---|---|---|---|
Human | Tested | Tested | Tested | Tested | Tested |
Mouse | Not recommended | Not recommended | Not recommended | Not recommended | Not recommended |
Rat | Not recommended | Not recommended | Not recommended | Not recommended | Not recommended |
Monkey | Not recommended | Not recommended | Not recommended | Not recommended | Not recommended |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/10 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat, Monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/200 | Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Species Rat | Dilution info - | Notes Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody. |
Species Monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/20 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Rat | Dilution info - | Notes - |
Species Monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/1000 - 1/10000 | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Mouse, Rat, Monkey | Dilution info - | Notes - |
Species | Dilution info | Notes |
---|---|---|
Species Human | Dilution info 1/50 - 1/250 | Notes For optimal performance in IHC, the primary antibody should be incubated overnight at 4°C. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species | Dilution info | Notes |
---|---|---|
Species Mouse | Dilution info - | Notes For optimal performance in IHC, the primary antibody should be incubated overnight at 4°C. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Rat | Dilution info - | Notes For optimal performance in IHC, the primary antibody should be incubated overnight at 4°C. Perform heat-mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
Species Monkey | Dilution info - | Notes - |
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B-lymphocyte-specific membrane protein that plays a role in the regulation of cellular calcium influx necessary for the development, differentiation, and activation of B-lymphocytes (PubMed:12920111, PubMed:3925015, PubMed:7684739). Functions as a store-operated calcium (SOC) channel component promoting calcium influx after activation by the B-cell receptor/BCR (PubMed:12920111, PubMed:18474602, PubMed:7684739).
CD20, MS4A1, CD20, B-lymphocyte antigen CD20, B-lymphocyte surface antigen B1, Bp35, Leukocyte surface antigen Leu-16, Membrane-spanning 4-domains subfamily A member 1
Anti-CD20 antibody [EP459Y] ab78237 is a rabbit monoclonal antibody that is used in CD20 western blotting, IHC, immunofluorescence and flow cytometry. Suitable for human samples.
- Recombinant format for unrivaled batch-batch consistency: no need for same-lot requests
- Antibody clone EP459Y has been tried and trusted by researchers since 2009 and is cited in >100 publications
- Specificity confirmed with MS4A1 knockout cell line validation
- Specificity and sensitivity confirmed in IHC with multi-tissue microarray (TMA) validation
IgG
Rabbit
pH: 7.2 - 7.4
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA
Liquid
Monoclonal
EP459Y
Affinity purification Protein A
For optimal performance in IHC, the primary antibody should be incubated overnight at 4°C.
Blue Ice
1-2 weeks
+4°C
-20°C
Upon delivery aliquot
Avoid freeze / thaw cycle
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
This product is a recombinant monoclonal antibody, which offers several advantages including:
For more information, read more on recombinant antibodies.
This supplementary information is collated from multiple sources and compiled automatically.
CD20 also known as MS4A1 or L26 is a membrane-spanning 4-domains subfamily A member 1 protein. It is a non-glycosylated phosphoprotein with a molecular mass of approximately 33-37 kDa. Expressed extensively on the surface of B-cells CD20 plays an important role in B-cell activation and regulation. While CD20 is absent on early pro-B cells and plasma cells its presence increases as B-cells mature. Anti-CD20 antibodies such as 2H7 are commonly used in research and treatment to deplete B-cells due to the target's consistent expression in most stages of B-cell development.
CD20 contributes significantly to calcium ion transport which is essential for the activation process of B-cells. Although CD20 does not belong to a larger protein complex its role centers around forming a calcium channel that allows a sustained influx of calcium into the B-cells. This influx is important for the signaling pathways that govern B-cell activation proliferation and differentiation impacting the immune response efficacy.
CD20 is closely involved in the B-cell receptor (BCR) signaling pathway and the Fc gamma R-mediated phagocytosis pathway. These pathways play vital roles in adaptive immunity and immune response modulation. CD20's interaction with proteins like PI3K during BCR signaling enhances receptor-mediated cellular signals subsequently influencing downstream effectors involved in cell growth and survival of B-cells.
CD20 is strongly linked to certain blood cancers and autoimmune diseases including non-Hodgkin's lymphoma and rheumatoid arthritis. These conditions often exploit aberrant B-cell activity or count. CD20's expression on malignant B-cells in non-Hodgkin's lymphoma makes it an effective target for monoclonal antibody therapies such as rituximab an anti-CD20 antibody. Additionally CD20-targeted therapies can help deplete pathogenic B-cells in rheumatoid arthritis highlighting the protein's relevance in disease treatment.
We have tested this species and application combination and it works. It is covered by our product promise.
We have not tested this specific species and application combination in-house, but expect it will work. It is covered by our product promise.
This species and application combination has not been tested, but we predict it will work based on strong homology. However, this combination is not covered by our product promise.
We do not recommend this combination. It is not covered by our product promise.
We are dedicated to supporting your work with high quality reagents and we are here for you every step of the way should you need us.
In the unlikely event of one of our products not working as expected, you are covered by our product promise.
Full details and terms and conditions can be found here:
Terms & Conditions.
False colour image of Western blot: Anti-CD20 antibody [EP459Y] - Rat IgG2a (Chimeric) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-CD20 antibody [EP459Y] - Rat IgG2a (Chimeric) 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 Human MS4A1 (CD20) knockout Raji cell line ab273871 (knockout cell lysate Human MRPS28 knockout HEK-293T 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 (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
Lanes 1 - 4: Western blot - Anti-CD20 antibody [EP459Y] (ab78237) at 1/1000 dilution
Lanes 1 - 4: Western blot - Anti-CD20 antibody [EP459Y] - Rat IgG2a (Chimeric) (Anti-CD20 antibody [EP459Y] - Rat IgG2a (Chimeric) ab279300) at 1/1000 dilution
Lane 1: Wild-type Raji cell lysate at 20 µg
Lane 2: MS4A1 knockout Raji cell lysate at 20 µg
Lane 3: A549 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 21 kDa, 33 kDa
Observed band size: 33 kDa
False colour image of Western blot: Anti-CD20 antibody [EP459Y] – Mouse IgG2a (Chimeric) staining at 1/1000 dilution, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) ab279299 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 Human MS4A1 (CD20) knockout Raji cell line ab273871 (knockout cell lysate Human MRPS28 knockout HEK-293T 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
Lanes 1 - 4: Western blot - Anti-CD20 antibody [EP459Y] (ab78237) at 1/1000 dilution
Lanes 1 - 4: Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) (Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) ab279299) at 1/1000 dilution
Lane 1: Wild-type Raji cell lysate at 20 µg
Lane 2: MS4A1 knockout Raji cell lysate at 20 µg
Lane 3: A549 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 21 kDa, 33 kDa
Observed band size: 33 kDa
ab78237 (purified) at 1/20 immunoprecipitating CD20 in 10 μg Ramos (Human Burkitt's lymphoma cell line) cell lysate (Lanes 1 and 2, observed at 33 kDa). Lane 3 - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730). For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10,000 dilution.
Blocking/Dilution buffer and concentration: 5% NFDM/TBST.
All lanes: Immunoprecipitation - Anti-CD20 antibody [EP459Y] (ab78237)
Predicted band size: 33 kDa
Immunocytochemistry/ Immunofluorescence analysis of Ramos (human Burkitt's lymphoma B lymphocyte) labeling CD20 with purified ab78237 at 1/10 dilution. Goat anti rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 was used as the secondary antibody. Cells were counterstained with Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889, Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) at 1/200. PBS instead of the primary antibody was used as negative control. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. Nuclei were stained with DAPI (blue)
Formaldehyde-fixed, paraffin-embedded human spleen tissue stained for CD20 using ab78237 at 1/50 dilution in immunohistochemical analysis.
Heat mediated antigen retieval with citrate pH 6.0.
Immunohistochemical staining of paraffin embedded human B cell lymphoma with purified ab78237 at a working dilution of 1/50.
The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counterstained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
PBS was used instead of the primary antibody as the negative control, and is shown in the inset.
Intracellular Flow Cytometry analysis ofRamos (Human Burkitt's lymphoma B lymphocyte) labeling CD20 with purified ab78237 at 1/200 dilution (Red). Goat anti rabbit IgG (Alexa Fluorr® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution was used as secondary antibody. Cells were fixed with 4% paraformaldehyde and permeabilized with 90% methanol.
Isotype control: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black)
Unlabelled cells: (Blue)
False colour image of Western blot: Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) staining, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (Anti-alpha Tubulin antibody [EP1332Y] - Microtubule Marker ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) ab279298 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 Human MS4A1 (CD20) knockout Raji cell line ab273871 (knockout cell lysate Human MRPS28 knockout HEK-293T 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-Mouse IgG H&L (IRDye® 800CW) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) at 1/20000 dilution.
Lanes 1 - 4: Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) ab279298) at 1/1000 dilution
Lanes 1 - 4: Western blot - Anti-CD20 antibody [EP459Y] (ab78237) at 1/1000 dilution
Lane 1: Wild-type Raji cell lysate at 20 µg
Lane 2: MS4A1 knockout Raji cell lysate at 20 µg
Lane 3: A549 cell lysate at 20 µg
Lane 4: HeLa cell lysate at 20 µg
Performed under reducing conditions.
Predicted band size: 21 kDa, 33 kDa
Observed band size: 33 kDa
Blocking/Dilution buffer: 5% NFDM/TBST.
All lanes: Western blot - Anti-CD20 antibody [EP459Y] (ab78237) at 1/10000 dilution
All lanes: Raji (Human Burkitt's lymphoma cell line) whole cell lysate
All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/20000 dilution
Predicted band size: 33 kDa
Observed band size: 33 kDa
Blocking/Dilution buffer: 5% NFDM/TBST.
Anti-CD20 antibody [EP459Y] - BSA and Azide free ab214282 is a carrier-free buffer format of ab78237. Please refer to ab78237 for recommended dilutions, protocols and image data.
Unpurified ab78237 staining human CD20 in human lymphoma tissue by immunohistochemistry using formalin fixed, paraffin embedded tissue.
All lanes: Western blot - Anti-CD20 antibody [EP459Y] (ab78237) at 1/5000 dilution
All lanes: Raji (Human Burkitt's lymphoma cell line) cell lysate
Predicted band size: 33 kDa
Observed band size: 33 kDa
Unpurified ab78237 showing positive staining in human spleen tissue.
Unpurified ab78237 showing negative staining in human brain tissue.
Unpurified ab78237 showing negative staining in human heart tissue.
Unpurified ab78237 showing negative staining in human kidney tissue.
Unpurified ab78237 showing positive staining in human tonsil tissue.
Flow cytometry overlay histogram showing left Ramos positive cells and right negative HEK293 stained with ab78237 (red line). The cells were fixed with 4% formaldehyde (10 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10μg/ml human IgG and 10% normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody (ab78237) (1x 106 in 100μl at 0.2μg/ml (1/2500)) for 30min at 22°C.
The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C
Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control 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 Fixed with 80% methanol (5 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.
Immunohistochemical analysis of formalin fixed paraffin embedded human spleen labelling CD20 with ab78237 at a concentration of 1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH 8.5 for 32mins.
ab78237 anti-CD20 [EP459Y] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.
Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).
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