Anti-ZAP70 antibody [E267]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ZAP70 antibody [E267] (AB32410)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (human T cell leukemia T lymphocyte from peripheral blood) labeling ZAP70 with ab32410 at 1/100 dilution followed by ab150081 at 1/1000 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. ab195889 at 1/200 was used as counterstain antibody. Nuclear counterstain was DAPI. Confocal image showing positive staining in Jurkat cell line (shown in green). The counterstain was observed with red. Nuclear DNA was labeled with DAPI (shown in blue). Negative control : Raji. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-ZAP70 antibody [E267] (AB32410)

Immunocytochemistry/ Immunofluorescence analysis of EL4 (mouse lymphoma T lymphocyte) labeling ZAP70 with ab32410 at 1/100 dilution followed by ab150081 at 1/1000 dilution. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. ab195889 at 1/200 was used as counterstain antibody. Nuclear counterstain was DAPI. Confocal image showing positive staining in El4 cell line (shown in green). The counterstain was observed with red. Nuclear DNA was labeled with DAPI (shown in blue). Negative control : A20. Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8).

• WB

Unknown

Western blot - Anti-ZAP70 antibody [E267] (AB32410)

Western blot analysis on Jurkat Cell lysate.

All lanes:

Western blot - Anti-ZAP70 antibody [E267] (ab32410) at 1/500 dilution

All lanes:

Jurkat cell lysates

Predicted band size: 69 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-ZAP70 antibody [E267] (AB32410)

False colour image of Western blot : Anti-ZAP70 antibody [E267] staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32410 was shown to bind specifically to ZAP70. A band was observed at 70 kDa in wild-type Jurkat cell lysates with no signal observed at this size in ZAP70 knockout cell line ab273841 (knockout cell lysate ab273795). The band observed in the knockout lysate lane below 70 kDa is likely to represent a truncated form of ZAP70. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZAP70 knockout Jurkat 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-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ZAP70 antibody [E267] (ab32410) at 1/500 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

ZAP70 knockout Jurkat cell lysate at 20 µg

Lane 3:

MOLT-4 cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Predicted band size: 69 kDa

Observed band size: 70 kDa

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• WB

Unknown

Western blot - Anti-ZAP70 antibody [E267] (AB32410)

Anti-GAPDH antibody, ab8245 (1/20000) was used as a primary antibody for the loading control and Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed, ab216776 (1/10000) was used as a loading control secondary antibody.

Lanes 1-2 : Merged signal (red and green). Green – ab32410 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa.
ab32410 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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 10000 dilution for 1 hour at room temperature before imaging.

The expression profile observed in Raji is consistent with the literature (PMID : 25275600).

Negative control : Raji (PMID : 25275600)

All lanes:

Western blot - Anti-ZAP70 antibody [E267] (ab32410) at 1/1000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

Raji (Human Burkitt's lymphoma B lymphocyte) whole 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: 69 kDa

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• WB

Lab

Western blot - Anti-ZAP70 antibody [E267] (AB32410)

False colour image of Western blot : Anti-ZAP70 antibody [E267] staining at 1/500 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab32410 was shown to bind specifically to ZAP70. A band was observed at 70 kDa in wild-type Jurkat cell lysates with no signal observed at this size in ZAP70 CRISPR-Cas9 edited cell line ab273841 (CRISPR-Cas9 edited cell lysate ab273795). The band observed in the CRISPR-Cas9 edited lysate lane below 70 kDa is likely to represent a truncated form of ZAP70. This has not been investigated further and the functional properties of the gene product have not been determined. To generate this image, wild-type and ZAP70 CRISPR-Cas9 edited Jurkat 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-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-ZAP70 antibody [E267] (ab32410) at 1/500 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

ZAP70 CRISPR-Cas9 edited Jurkat cell lysate at 20 µg

Lane 2:

Western blot - Human ZAP70 knockout Jurkat cell line (<a href='/en-us/products/cell-lines/human-zap70-knockout-jurkat-cell-line-ab273841'>ab273841</a>)

Lane 3:

MOLT-4 cell lysate at 20 µg

Lane 4:

Raji cell lysate at 20 µg

Predicted band size: 69 kDa

Observed band size: 70 kDa

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