Anti-CD20 antibody [EP459Y] - BSA and Azide free

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

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, ab150077) at 1/1000 was used as the secondary antibody. Cells were counterstained with 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Unpurified ab78237 showing positive staining in human spleen tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Unpurified ab78237 showing negative staining in human kidney tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Unpurified ab78237 staining human CD20 in human lymphoma tissue by immunohistochemistry using formalin fixed, paraffin embedded tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Unpurified ab78237 showing negative staining in human brain tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Unpurified ab78237 showing negative staining in human heart tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Unpurified ab78237 showing positive staining in human tonsil tissue.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

Immunohistochemical staining of paraffin embedded human B cell lymphoma with purified ab78237 at a working dilution of 1/50.

The secondary antibody used is 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

• IP

Unknown

Immunoprecipitation - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

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 (ab172730). For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10,000 dilution.

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).

All lanes:

Immunoprecipitation - Anti-CD20 antibody [EP459Y] (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-ab78237'>ab78237</a>)

Predicted band size: 33 kDa

false

• WB

Lab

Western blot - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

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] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, 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 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 4:

Western blot - Anti-CD20 antibody [EP459Y] (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-ab78237'>ab78237</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-mouse-igg2a-chimeric-ab279299'>ab279299</a>) 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 2:

Western blot - Human MS4A1 (CD20) knockout Raji cell line (<a href='/en-us/products/cell-lines/human-ms4a1-cd20-knockout-raji-cell-line-ab273871'>ab273871</a>)

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 21 kDa,33 kDa

Observed band size: 33 kDa

false

• WB

Lab

Western blot - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

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] (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.

Lanes 1 - 4:

Western blot - Anti-CD20 antibody [EP459Y] (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-ab78237'>ab78237</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-CD20 antibody [EP459Y] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-rat-igg2a-chimeric-ab279300'>ab279300</a>) 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 2:

Western blot - Human MS4A1 (CD20) knockout Raji cell line (<a href='/en-us/products/cell-lines/human-ms4a1-cd20-knockout-raji-cell-line-ab273871'>ab273871</a>)

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 21 kDa,33 kDa

Observed band size: 33 kDa

false

• WB

Lab

Western blot - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

False colour image of Western blot : Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) staining, shown in green; Rabbit anti-alpha Tubulin antibody [EP1332Y] (ab52866) loading control staining at 1/20000 dilution, shown in red. In Western blot, 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 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

Lanes 1 - 4:

Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-mouse-igg1-chimeric-ab279298'>ab279298</a>) at 1/1000 dilution

Lanes 1 - 4:

Western blot - Anti-CD20 antibody [EP459Y] (<a href='/en-us/products/primary-antibodies/cd20-antibody-ep459y-ab78237'>ab78237</a>) 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 2:

Western blot - Human MS4A1 (CD20) knockout Raji cell line (<a href='/en-us/products/cell-lines/human-ms4a1-cd20-knockout-raji-cell-line-ab273871'>ab273871</a>)

Lane 3:

A549 cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 21 kDa,33 kDa

Observed band size: 33 kDa

false

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD20 antibody [EP459Y] - BSA and Azide free (AB214282)

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 Fluor^® 488, 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 (ab172730) (Black)

Unlabelled cells : (Blue)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab78237).