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Total ZAP-70 cellular kit HTRF®

The total ZAP-70 kit detects cellular ZAP-70 and can be combined with our phospho-ZAP-70 kit for an optimal readout of the Hippo-YAP pathway.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
The total ZAP-70 kit detects cellular ZAP-70 and can be combined with our phospho-ZAP-70 kit for an optimal readout of the Hippo-YAP pathway.
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Overview

The Total ZAP-70 cellular assay is designed to monitor the expression level of ZAP-70 proteins, both phosphorylated and unphosphorylated. It is compatible with our phospho-ZAP-70 kit and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of T-cell activation. ZAP-70 is a cytoplasmic protein tyrosine kinase expressed in T and NK cells, and plays a critical role in the events involved in initiating T-cell responses by the antigen receptor.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-ZAP-70 assay principle

The Total-ZAP-70 assay quantifies the expression level of ZAP-70 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-ZAP-70 assay uses two labeled antibodies: one coupled to a donor fluorophore, the other to an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of ZAP-70 in a cell extract, the addition of these conjugates brings the donor fluorophore into close proximity with the acceptor and thereby generates a FRET signal. Its intensity is directly proportional to the concentration of the protein present in the sample, and provides a means of assessing the protein’s expression under a no-wash assay format.

Total-ZAP-70 assay principle

Total-ZAP-70 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis then transferring lysates to a 384-well low volume detection plate before adding Total-ZAP-70 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-ZAP-70 2-plate assay protocol

Total-ZAP-70 1-plate assay protocol

Detection of total ZAP70 with HTRF reagents can be performed in a single plate used for culturing, stimulation and lysis. No washing steps are required. This HTS designed protocol enables miniaturization while maintaining robust HTRF quality.
Total-ZAP-70 1-plate assay protocol

HTRF assay compared to Western Blot using ZAP-70 cellular assays on human Jurkat cells

Jurkat cells were grown in a T175cm2 flask for 2 days in RPMI culture medium supplemented by 10% FBS, at 37°C in 5% CO2 atmosphere. 4.5 ml of cell suspension (13 x 106 cells / mL) were stimulated with anti-CD3 antibody (20 µg / mL final concentration during 2.5min). After stimulation, cells were lysed at RT for 30 min by adding 2.25 mL of 4X supplemented lysis buffer. Neat lysate was then serially diluted in the same supplemented lysis buffer. 16 µL of each dilution were analyzed in parallel by HTRF or Western Blot
HTRF assay compared to Western Blot using ZAP-70 cellular assays on human Jurkat cells

Kinetics of cell stimulation - Total ZAP-70 assay

This assay was performed using the two-plate assay protocol. Jurkat cells were plated in a 96-well culture plate at 400 K cells, using 25 µL / well in complete RPMI culture medium with 10% FBS. Cell stimulation was performed using the anti-CD3 Ab at the final concentration of 20 µg / mL for different stimulation times (min). After stimulation, cells were lysed with 10 µL of 4X supplemented lysis buffer. 16 µl of each cell lysate type were analyzed with the HTRF total ZAP-70 assay.
Kinetics of cell stimulation - Total ZAP-70 assay

Monitoring of the immune checkpoint inhibitor PD-L1 activity

As Phospho ZAP-70 (Tyr319) is a readout of T cell activation, it was used to monitor the inhibitory effect of PD-L1 recombinant protein on T cell activation. This assay was performed using the two-plate assay protocol. 400 000 Jurkat cells were plated in 25 µL of complete RPMI culture medium with 10% FBS in a 96-well culture plate. Cells were pre-incubated 24 hours with different concentrations of PD-L1 recombinant protein. Cells were then stimulated for 2.5 min with 20 µg / mL anti-CD3 Ab (final concentration) and lysed directly with10 µL of 4X supplemented lysis buffer, then incubated 30 min at RT. 16µL of each sample were analyzed using Phospho ZAP-70 (Tyr319) and Total ZAP-70 assays. Phospho ZAP-70 results were normalized with the Total ZAP-70 amount and plotted on a graph. A slight but significant inhibitory effect of PD-L1 recombinant protein was recorded with the Phospho ZAP-70 (Tyr319) assay at the final concentration of 40 µg / mL.
Monitoring of the immune checkpoint inhibitor PD-L1 activity

Function and regulation of ZAP-70

TCR engagement promotes the phosphorylation of immunoreceptor tyrosine-based activation motifs (ITAMs) on the cytosolic side of the TCR/CD3 complex by lymphocyte protein tyrosine kinase (Lck). Zap-70 is recruited to the TCR/CD3 complex where it becomes phosphorylated and activated, promoting recruitment and phosphorylation of downstream adaptor or scaffold proteins.
Function and regulation of ZAP-70, pathway representation

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF assays for Oncology and Inflammation

Signaling in the immune system - Brochures

Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Application Notes

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Videos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Videos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Application Notes

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.

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