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

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.

Blocking/Dilution buffer: 5% NFDM/TBST.

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 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.

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.

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.