All lanes : Anti-MALT1/MLT antibody [EP603Y] (ab33921) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : MALT1 knockout HeLa cell lysate
Lane 3 : Ramos cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (ab216773) at 1/10000 dilution

Predicted band size: 92 kDa
Observed band size: 92 kDa

Lanes 1-3: Merged signal (red and green). Green - ab33921 observed at 92 kDa. Red - loading control ab7291 observed at 50 kDa.

ab33921 Anti-MALT1/MLT antibody [EP603Y] was shown to specifically react with MALT1/MLT in wild-type HeLa cells. Loss of signal was observed when knockout cell line ab264930 (knockout cell lysate ab257149) was used. Wild-type and MALT1/MLT knockout samples were subjected to SDS-PAGE. ab33921 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

 

Immunocytochemistry/Immunofluorescence analysis of Ramos cells labelling MALT1/MLT with purified ab33921 at 1/250. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/500) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500) were also used.

Control 1: Primary antibody (1/100) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/500).

Control 2: ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/500).

All lanes : Anti-MALT1/MLT antibody [EP603Y] (ab33921) at 1/10000 dilution (purified)

Lane 1 : Ramos (Human Burkitt's lymphoma cell line) cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : K562 (Human chronic myelogenous leukemia cell line from bone marrow) cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Peroxidase conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 92 kDa
Observed band size: 92 kDa

Blocking and dilution buffer: 5% NFDM/TBST.

Flow Cytometry analysis of Jurkat (Human T cell leukemia cell line from peripheral blood) cells labelling MALT1/MLT with purified ab33921 at 1/100 (red). Cells were fixed with 80% methanol. A FITC-conjugated goat anti-rabbit IgG (1/150) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

Anti-MALT1/MLT antibody [EP603Y] (ab33921) at 1/2000 dilution (unpurified) + Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate

Predicted band size: 92 kDa
Observed band size: 92 kDa

Overlay histogram showing Jurkat (Human T cell leukemia cell line from peripheral blood) cells stained with unpurified ab33921 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (unpurified ab33921, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

This antibody gave a positive signal in Jurkat cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.