Recombinant Anti-ZAP70 antibody [YE291] (ab32429)

All lanes : Anti-ZAP70 antibody [YE291] (ab32429) at 1/500 dilution

Lane 1 : Wild-type Jurkat cell lysate
Lane 2 : ZAP70 CRISPR-Cas9 edited Jurkat cell lysate
Lane 3 : MOLT-4 cell lysate
Lane 4 : Raji cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 70 kDa
Observed band size: 70 kDa

False colour image of Western blot: Anti-ZAP70 antibody [YE291] 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, ab32429 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.

ZAP70 was immunoprecipitated from 0.35 mg Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 µg with ab32429 at 1/50 dilution (2µg). VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate 10 µg

Lane 2: ab32429 IP in Jurkat whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab32429 in Jurkat whole cell lysate

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

All lanes : Anti-ZAP70 antibody [YE291] (ab32429) at 1/1000 dilution

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate
Lane 2 : Raji (Human Burkitt's lymphoma B lymphocyte) whole 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 (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed)

Predicted band size: 70 kDa

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 – ab32429 observed at 70 kDa. Red - loading control ab8245 observed at 36 kDa.
 ab32429 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)

Ab32429, at a 1/100 dilution, staining ZAP70 in paraffin embedded human lymph node tissue by immunohistochemistry.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Anti-ZAP70 antibody [YE291] (ab32429) at 1/500 dilution + Jurkat cell lysate

Predicted band size: 70 kDa
Observed band size: 70 kDa

Intracellular Intracellular Intracellular Flow Cytometry analysis of Jurkat cells labeling ZAP70 with ab32429 at 1/80 dilution (red) or rabbit IgG as negative control (green). 

 

 

ab32429 showing positive staining in Normal tonsil tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32429 showing negative staining in Normal liver tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32429 showing negative staining in Normal heart tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

ab32429 showing negative staining in Normal kidney tissue.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.