Anti-MALT1/MLT antibody [EP603Y]

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

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 Fluorr^® 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

Intracellular Flow Cytometry analysis ofJurkat (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

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).

• IP

Lab

Immunoprecipitation - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

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] (ab33921)

Predicted band size: 92 kDa

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Exposure time: 32s

• WB

Lab

Western blot - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

Lanes 1-4 : 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 sample 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 (IRDye800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye680RD) 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] (ab33921) 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

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Western blot - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-MALT1/MLT antibody [EP603Y] (ab33921) at 1/10000 dilution

Lane 1:

Ramos (Human Burkitt's lymphoma cell line) cell lysate at 10 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate at 10 µg

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) cell lysate at 10 µg

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

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• WB

Lab

Western blot - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

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] (ab33921) 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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• WB

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Western blot - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

All lanes:

Western blot - Anti-MALT1/MLT antibody [EP603Y] (ab33921) at 1/2000 dilution

All lanes:

Jurkat (Human T cell leukemia cell line from peripheral blood) cell lysate

Predicted band size: 92 kDa

Observed band size: 92 kDa

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• WB

CiteAb

Western blot - Anti-MALT1/MLT antibody [EP603Y] (AB33921)

MALT1/MLT western blot using anti-MALT1/MLT antibody [EP603Y] ab33921. Publication image and figure legend from Ginster, S., Bardet, M., et al., 2017, PLoS One, PubMed 28052131.

ab33921 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab33921 please see the product overview.

Ectopic CBM reconstitution triggers MALT1 auto-cleavage and ubiquitination.(A) Constituents of the CBM complex ─ CARD11-L244P, BCL10 and MALT1 (N-terminal FLAG) ─ were ectopically expressed in HEK293 cells individually or in combination (CBM reconstitution assay). 24h after transfection, lysates were analyzed with anti-CARD11, anti-BCL10 (ep605y), anti-MALT1 (ep603y) and anti-FLAG antibodies. BCL10 cleaved by MALT1 is indicated with Δ5. Phosphorylated BCL10 species are indicated with (P). The white arrow head points to a faster migrating species of MALT1. (B) CBM reconstitution was performed in the presence of z-VRPR-fmk, a covalent peptidic inhibitor of MALT1 protease (10) or by using a catalytically deficient MALT1-C464A mutant form. The white arrow head points to a faster migrating species of MALT1, the black one to a slower running species. (C) MALT1A-824 (WT), R781A, 1–781 and R800A expression constructs were submitted to a CBM reconstitution assay and analyzed by Western Blot with an anti-FLAG antibody to detect MALT1. This identified the main self-cleavage product (white arrow) to result from cleavage at R781. (D) MALT1A-824 WT, C464A and C464A/K644R expression constructs were submitted to a CBM reconstitution assay and analyzed by Western Blot with an anti-FLAG antibody to detect MALT1. This showed that the slower migrating species of MALT1 in our experiments (black arrow) corresponds to the K644-monoubiquitinated MALT1 species identified by Thome and coll. [19]. (E) MALT1A and MALT1B both undergo C-terminal cleavage and ubiquitination. CBM reconstitution was performed with MALT1A-824, R781-cleaved MALT1A (MALT1A-781), MALT1B-813 and R770-cleaved MALT1B (MALT1B-770), in the absence or presence of z-VRPR-fmk. The white arrow head points to faster migrating species of MALT1A and B, the black one to a slower running species of these two isoforms.

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