Anti-ZAP70 antibody [29/ZAP70 Kinase] (BSA and Azide free)

• WB

Supplier Data

Western blot - Anti-ZAP70 antibody [29/ZAP70 Kinase] (BSA and Azide free) (AB302822)

This data was developed using ab302821, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBSLysates at 20 µg per lane. False colour image of Western blot : Anti-ZAP70 antibody[29/ZAP70 Kinase] staining at 1/1000 dilution, shown in green; Rabbit anti-GAPDH antibody EPR16891 loading control staining at 1/20000 dilution, shown in red. In Western blot, ab302821 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 the ZAP70 knockout cell line. 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 PVDF-FL membrane. Membranes were blocked in Odyssey diluted in equal volume of 0.1 % TBS 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-Mouse IgG H&L (IRDye® 800CW) (ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) (ab216777) at 1/10000 dilution. Performed under reducing conditions.

All lanes:

Western blot - Anti-ZAP70 antibody [29/ZAP70 Kinase] (<a href='/en-us/products/primary-antibodies/zap70-antibody-29-zap70-kinase-ab302821'>ab302821</a>) at 1/1000 dilution

Lane 1:

Wild-type Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 2:

ZAP70 knockout Jurkat whole cell lysate at 20 µg

Secondary

Lanes 1 - 2:

Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-800cw-preadsorbed-ab216772'>ab216772</a>) at 1/10000 dilution

Lanes 1 - 2:

Western blot - Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-680rd-preadsorbed-ab216777'>ab216777</a>) at 1/10000 dilution

Observed band size: 70 kDa

false

• WB

Supplier Data

Western blot - Anti-ZAP70 antibody [29/ZAP70 Kinase] (BSA and Azide free) (AB302822)

This data was developed using ab302821, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. Negative control : Raji (PMID : 25275600), Ramos(PMID : 21865343). The identity of the higher Mw band at approximately 75 kDa is unknown. Exposure time : 26 seconds

All lanes:

Western blot - Anti-ZAP70 antibody [29/ZAP70 Kinase] (<a href='/en-us/products/primary-antibodies/zap70-antibody-29-zap70-kinase-ab302821'>ab302821</a>) at 1/1000 dilution

Lane 1:

H9 (human lymphoma cutaneous T lymphocyte), whole cell lysate at 20 µg

Lane 2:

Raji (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Lane 3:

Ramos (human Burkitt's lymphoma B lymphocyte), whole cell lysate at 20 µg

Lane 4:

EL4 (mouse lymphoma T lymphocyte), whole cell lysate at 20 µg

Lane 5:

A20 (mouse reticulum sarcoma B lymphocyte), whole cell lysate at 20 µg

Observed band size: 70 kDa

false

Exposure time: 26s