Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free (AB239823)

Overlay histogram showing Jurkat 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33921).

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free (AB239823)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33921).

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free (AB239823)

Intracellular Flow Cytometry analysis ofJurkat 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab33921).

• WB

Lab

Western blot - Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free (AB239823)

This data was developed using the same antibody clone in a different buffer formulation (ab33921).

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.

All lanes:

Western blot - Anti-MALT1/MLT antibody [EP603Y] (<a href='/en-us/products/primary-antibodies/malt1-mlt-antibody-ep603y-ab33921'>ab33921</a>) at 1/1000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

MALT1 knockout HeLa cell lysate at 20 µg

Lane 2:

Western blot - Human MALT1 (MLT) knockout HeLa cell line (<a href='/en-us/products/cell-lines/human-malt1-mlt-knockout-hela-cell-line-ab264930'>ab264930</a>)

Lane 3:

Ramos cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 92 kDa

Observed band size: 92 kDa

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• WB

Lab

Western blot - Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free (AB239823)

This data was developed using ab33921, the same antibody clone in a different buffer formulation.

Western blot : Rabbit Monoclonal[EP603Y] to MALT1/MLT ab33921 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at kDa in Wild-type HCT 116 ab288559 cell lysates with no signal observed at this size in MALT1 knockout HCT 116 ab286597 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc 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-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MALT1/MLT antibody [EP603Y] (<a href='/en-us/products/primary-antibodies/malt1-mlt-antibody-ep603y-ab33921'>ab33921</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 ab288559 at 20 µg

Lane 2:

Western blot - Human MALT1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-malt1-knockout-hct116-cell-line-ab286597'>ab286597</a>) at 20 µg

Lane 3:

HeLa at 20 µg

Lane 4:

Ramos at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 95 kDa

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• IP

Lab

Immunoprecipitation - Anti-MALT1/MLT antibody [EP603Y] - BSA and Azide free (AB239823)

This data was produced using ab33921, the same clone but in a different formulation.

MALT1/MLT was immunoprecipitated from 0.35 mg of Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab33921 at 1/50 dilution. Western blot was performed from the immunoprecipitate using ab33921 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/1000 dilution.

Lane 1 : Ramos whole cell lysate 10 μg (Input).

Lane 2 : ab33921 IP in Ramos whole cell lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab33921 in Ramos whole cell lysate.

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

All lanes:

Immunoprecipitation - Anti-MALT1/MLT antibody [EP603Y] (<a href='/en-us/products/primary-antibodies/malt1-mlt-antibody-ep603y-ab33921'>ab33921</a>)

Predicted band size: 92 kDa

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Exposure time: 32s