重组Anti-ZAP70抗体[YE291]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] (AB32429)

ab32429 showing negative staining in Normal liver tissue.

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

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] (AB32429)

ab32429 showing positive staining in Normal tonsil tissue.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] (AB32429)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab32429 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab32429 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.04 μg/ml (1/50000)) for 30 min at 22°C. The cells were simultaneously stained with CD19.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C.

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] (AB32429)

ab32429 showing negative staining in Normal kidney tissue.

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

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] (AB32429)

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).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] (AB32429)

Flow cytometry staining of human peripheral blood mononuclear cells (PBMCs) with ab32429 (right) or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (left). Cells were fixed and permeabilised with BD Cytofix/Cytoperm™ for 20 min. PBMCs were incubated for 30 min on ice in 1x PBS containing 10 µg/ml human IgG and 10 % normal goat serum to block FC receptors and non-specific protein-protein interaction followed by the antibody ab32429 or Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control (1x 10⁶ in 100 µl at 0.04 μg/ml (1/50000)) for 30 min at 22°C . The cells were simultaneously stained with CD19.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Acquisition of >30000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter. Events were gated on live cells.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] (AB32429)

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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] (AB32429)

ab32429 showing negative staining in Normal heart tissue.

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

• IP

Lab

Immunoprecipitation - Anti-ZAP70 antibody [YE291] (AB32429)

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:

Immunoprecipitation - Anti-ZAP70 antibody [YE291] (ab32429)

Predicted band size: 69 kDa

Observed band size: 70 kDa

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• WB

Lab

Western blot - Anti-ZAP70 antibody [YE291] (AB32429)

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 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 [YE291] (ab32429) 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

false

• WB

Lab

Western blot - Anti-ZAP70 antibody [YE291] (AB32429)

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.

All lanes:

Western blot - Anti-ZAP70 antibody [YE291] (ab32429) 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='/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

false

• WB

Unknown

Western blot - Anti-ZAP70 antibody [YE291] (AB32429)

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)

All lanes:

Western blot - Anti-ZAP70 antibody [YE291] (ab32429) 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='/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) at 1/10000 dilution

Predicted band size: 69 kDa

false

• WB

Unknown

Western blot - Anti-ZAP70 antibody [YE291] (AB32429)

All lanes:

Western blot - Anti-ZAP70 antibody [YE291] (ab32429) at 1/500 dilution

All lanes:

Jurkat cell lysate

Predicted band size: 69 kDa

Observed band size: 70 kDa

false

• WB

CiteAb

Western blot - Anti-ZAP70 antibody [YE291] (AB32429)

ZAP70 western blot using anti-ZAP70 antibody [YE291] ab32429. Publication image and figure legend from Ren, L., Li, P., et al., 2022, BMC Cancer, PubMed 35477402.

ab32429 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 ab32429 please see the product overview.

AQP9 and ZAP70 up-regulation inhibits cell viability of laryngeal cancer. A, B RT-qPCR for AQP9 and ZAP70 mRNA expression in TU212 and LCC cells transfected with siRNAs against AQP9 and ZAP70 or their overexpression plasmids. C-E Western blot for AQP9 and ZAP70 expression in TU212 and LCC cells transfected with siRNAs against AQP9 and ZAP70 or their overexpression plasmids. F, G CCK-8 for cell viability of TU212 and LCC cells transfected with si-AQP9 or AQP9 overexpression plasmid. H, I CCK-8 for cell viability of TU212 and LCC cells transfected with si-ZAP70 or ZAP70 overexpression plasmid. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

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