Anti-ZAP70 antibody [YE291] - BSA and Azide free

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

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

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32429).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (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.

• IP

Lab

Immunoprecipitation - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

This data was developed using ab32429, the same antibody clone in a different buffer formulation.

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] (<a href='/en-us/products/primary-antibodies/zap70-antibody-ye291-ab32429'>ab32429</a>)

Predicted band size: 69 kDa

Observed band size: 70 kDa

false

• WB

Lab

Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32429).

All lanes:

Western blot - Anti-ZAP70 antibody [YE291] (<a href='/en-us/products/primary-antibodies/zap70-antibody-ye291-ab32429'>ab32429</a>) 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

false

• WB

Unknown

Western blot - Anti-ZAP70 antibody [YE291] - BSA and Azide free (AB247258)

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)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32429).

All lanes:

Western blot - Anti-ZAP70 antibody [YE291] (<a href='/en-us/products/primary-antibodies/zap70-antibody-ye291-ab32429'>ab32429</a>) 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

false