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Total SHP1 cellular kit HTRF®

The Total SHP1 kit is designed to monitor the expression level of SHP1, and can be used as a normalization assay for the Phospho-SHP1 (Tyr564) kit.

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  • All inclusive kit All inclusive kit
  • High sensitivity High sensitivity
  • No-wash No-wash
  • Low sample consumption Low sample consumption

The Total SHP1 kit is designed to monitor the expression level of SHP1, and can be used as a normalization assay for the Phospho-SHP1 (Tyr564) kit.

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Overview

The Total SHP1 cellular assay monitors the expression level of SHP1, and can be used as a normalization assay with our phospho-SHP1 Y564 kit. Because these phospho and total SHP1 assays are compatible, the two kits can be used in parallel on the same lysates.

Many cancer cells overexpress checkpoint inhibitor ligands such as PD-L1. PD-L1 binds its counterpart checkpoint inhibitor receptor PD1, present at the surface of T lymphocytes. In turn, the PD1-PDL1 complex recruits and activates inhibitory effectors, such as SHP1 or SHP2. These two phosphatases, which are phosphorylated on Tyr564 and Tyr542 respectively by the kinase Lck, trigger the dephosphorylation of signaling proteins such as ZAP-70 or SLP-76, involved in the T cell activation pathway. Finally, activated SHP1 and SHP2 participate in T cell inactivation.

Preventing the activation of SHP1 and/or SHP2 by small molecule inhibitors is believed to contribute to restoring the immune response against tumors.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total SHP1 assay principle

The Total SHP1 assay measures the expression level of SHP1 independently of its phosphorylation state. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total SHP1 assay uses 2 labeled antibodies, one with a donor fluorophore, the other with an acceptor. Both antibodies are highly specific for a distinct epitope on the protein. In presence of SHP1 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-shp1-assay-principle

Total SHP1 two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total SHP1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

total-shp1-2-plate-assay-protocol

Total SHP1 one-plate assay protocol

Detection of Total SHP1 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-shp1-1-plate-assay-protocol

Pharmacological validation using the Lck inhibitor, saracatinib, in Jurkat T-cells

Human Jurkat suspension cells were plated at 100,000 cells/well in a 96-well half area plate, and incubated for 24 h at 37°C, 5% CO2, with increasing concentrations of Saracatinib. Before lysis, Jurkat cells were incubated 30min with Pervanadate (30 µM), followed by the addition of 10µL of supplemented lysis buffer 4X. After 30min lysis at RT under gentle shaking, 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-SHP1 (Tyr564) or total SHP1 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

As described elsewhere, a dose dependent inhibition of SHP1 Tyr564 phosphorylation was obtained following treatment with Saracatinib, whereas the SHP1 expression level remained stable under the same experimental conditions.

Image of the validation of the HTRF phospho and total SHP1 assays on human Jurkat cells treated with Saracatinib and Pervanadate

HTRF total-SHP1 cellular assays compared to Western Blot

The human Jurkat cell line was seeded in a T175 flask, and incubated at 37°C, 5% CO2. The cells were then treated with Pervanadate (30 µM) for 30 min before lysis.

Serial dilutions of the cell lysate were performed in the supplemented lysis buffer, and 16µL of each dilution were transferred into a low volume white microplate before the addition of 4µL of HTRF total-SHP1 detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

For HTRF total-SHP1 assay and for Western Blot, 5,000 cells/well were sufficient to detect a signal, demonstrating the similar sensitivity of the two methods.

Image of the comparison between Western blot and HTRF using total SHP1 on human Jurkat cells treated with Pervanadate

Function and regulation of SHP1

SHP1 (also known as tyrosine-protein phosphatase non-receptor type 6, PTPN6) is a tyrosine phosphatase mainly expressed in hematopoietic cells, activated by Lck and recruited by cellular surface receptors. SHP2 (also known as tyrosine-protein phosphatase non-receptor type 11) is ubiquitously expressed in hematopoietic or non-hematopoietic cells. Although SHP2 negatively regulates T cell activation, SHP2 is positively involved in ERK activation in response to growth factors such as PDGF or FGF.

In T lymphocyte cells, SHP1 and 2 are recruited by immune checkpoint inhibitors, thereby participating in the suppression of the TCR signaling pathway. SHP1 and 2 interact with PD1 ITIM domains and are phosphorylated and activated by the Lck kinase. Activated SHP1 and 2 phosphatases lead to the dephosphorylation of key TCR signaling effectors, such as ZAP70 or SLP76, which are required for T-cell proliferation and function.

Pathway total SHP1

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

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

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

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Properly interpret your compound effect - Application Notes

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Insider Tips for successful sample treatment - Technical Notes

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

Increased flexibility of phospho-assays - Application Notes

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

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

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

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Study a pathway of interest in PBMC and T cells - Application Notes

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

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

Investigating kinase activity in a cellular context

HTRF cellular assays - Scientific Presentations

Species compatibility

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

Inflammation cell by cell

HTRF solutions for each cell type - Flyers

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Physiologically relevant results fo fast flowing research - Flyers

Lysis buffer compatibility

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Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

STING HTRF offer to bridge innate and adaptive immunity

cGAS-STING signaling pathway from A to Z - Brochures

Product Insert SHP1 total Kit / 64NH1PEG-64NH1PEH

64NH1PEG-64NH1PEH - Product Insert

HTRF Product Catalog

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

Novel HTRF platform to delineate STING pathway

Explore the whole STING pathway with a single technology - Posters

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

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

An innate and adaptive immunity recap

Insight into the diversity of cells & signaling pathways - Guides

Advance your research on Fibrosis

Kits and reagents for Fibrosis research - Flyers

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

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