Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric)

• ICC

Lab

Immunocytochemistry - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (AB279298)

Immunofluorescence staining of CD20 using ab279298 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 ab279298 at 0.2 μg/ml. Cells were then incubated with ab150117, Goat Anti-Mouse 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 ab279298 but otherwise processed the same.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (AB279298)

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 ab279298 (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 (ab279298) (1x10⁶ in 100μl at 0.2 μg/ml) for 30 min at 22°C.

The secondary antibody Goat anti-mouse IgG H&L (Alexa Fluor^® 488, pre-adsorbed) (ab150177) was used at 1/2000 for 30 min at 22°C.

Isotype control antibody (black line) was mouse IgG1 kappa (ab170190) 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.

• ICC

Lab

Immunocytochemistry - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (AB279298)

Immunofluorescence staining of CD20 using ab279298 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 ab279298 at 0.2 μg/ml. Cells were then incubated with ab150117, Goat Anti-Mouse 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 ab279298 but otherwise processed the same.

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

• IP

Supplier Data

Immunoprecipitation - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (AB279298)

CD20 was immunoprecipitated from 0.35 mg Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate 10 μg with ab279298 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab279298 at 1/1000 dilution. Mouse IgG for IP (HRP) (ab131368) was used at 1/5000 dilution.

Lane 1 : Ramos whole cell lysate 10μg.

Lane 2 : ab279298 IP in Ramos whole cell lysate.

Lane 3 : Mouse monoclonal IgG1 (ab18443) instead of ab279298 in Ramos whole cell lysate.

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

Exposure time : 5.5 seconds.

All lanes:

Immunoprecipitation - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (ab279298)

Predicted band size: 33 kDa

false

• WB

Supplier Data

Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (AB279298)

Blocking/Dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (ab279298) at 1/1000 dilution

Lane 1:

Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Lane 2:

Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-Conjugated Goat anti-Mouse IgG (H+L) at 1/5000 dilution

Predicted band size: 33 kDa

false

• WB

Lab

Western blot - Anti-CD20 antibody [EP459Y] - Mouse IgG1 (Chimeric) (AB279298)

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) (ab279298) 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