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Phospho-PLCg2 (Tyr1217) Cellular Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated PLCg2 (Phospholipase C gamma 2) at Tyr1217, as a readout of multiple adaptive and innate immune cell surface receptors.

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

This HTRF kit enables the cell-based quantitative detection of phosphorylated PLCg2 (Phospholipase C gamma 2) at Tyr1217, as a readout of multiple adaptive and innate immune cell surface receptors.

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Overview

This HTRF cell-based assay conveniently and accurately detects phosphorylated Phospholipase C gamma 2 (PLCgamma 2) at Tyr1217.

PLCgamma 2 is a phosphoinositide-specific phospholipase that generates second messengers and plays a crucial role in signal transduction, especially in haematopoietic cells such as B cells, mast cells, macrophages, natural killer cells and platelets. Upon stimulation of different receptors such as B-cell antigen receptor (BCR) and Fc receptors (FcRs), PLCgamma 2 interacts with several adapter proteins and becomes phosphorylated by intracellular tyrosine kinases. Phosphorylation includes several sites, one of which is Tyr1217.

PLCgamma 2 has been linked to various pathologies such as auto-immune and inflammatory diseases, drug-resistance in cancer and neurodegeneration.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-PLCg2 (Tyr1217) assay principle

The Phospho-PLCg2 (Tyr1217) assay measures PLCg2 when phosphorylated at Tyr1217. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The assay uses 2 antibodies, one labeled with a donor fluorophore and the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both labeled antibodies, and which brings the donor fluorophore into close proximity to the acceptor, thereby generating a FRET signal. Its intensity is directly proportional to the concentration of phosphorylated protein present in the sample, and provides a means of assessing the protein's phosphorylation state under a no-wash assay format.

Principle of the HTRF phospho-PLCG2 (Tyr1217) assa

Phospho-PLCg2 (Tyr1217) two-plate assay protocol

The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before the addition of Phospho-PLCg2 (Tyr1217) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Two-plate protocol of the HTRF phospho-PLCG2 (Tyr1217) assay

Phospho-PLCg2 (Tyr1217) one-plate assay protocol

Detection of Phosphorylated PLCg2 (Tyr1217) 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.

One-plate protocol of the HTRF phospho-PLCG2 (Tyr1217) assay

PLCg2 activation in anti-IgM-stimulated Ramos B cells

The human B cell lymphoma Ramos cell line was seeded in a half area 96-well culture-treated plate at 400,000 cells/well in 25 µL complete culture medium. After 3 hour incubation  at 37 °C, 5% CO2, cells were stimulated with 5 µL of increasing concentrations of an anti-human IgM antibody for 5 minutes, and then lyzed with 10 µL of supplemented lysis buffer #4 (4X) for 30 minutes at RT under gentle shaking. For the detection step, 16 µL of cell lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF Phospho-PLCg2 (Tyr1217) or Total-PLCg2 detection reagents were added. The HTRF signal was recorded after an overnight incubation.

The anti-human IgM antibody induced a dose-dependent increase in PLCg2 phosphorylation at Tyr1217 without changing the expression level of the protein, demonstrating the activation of the BCR complex at the cell surface.

Validation of HTRF PLCg2 kits on Ramos cells

HTRF phospho-PLCg2 (Tyr1217) assay compared to western blot

The human B cell lymphoma Ramos cell line was cultured in a T175 flask in complete culture medium for 48h at 37 °C, 5% CO2. After centrifugation, pelleted cells were stimulated with 20 µg/mL of an anti-human IgM antibody for 5 minutes. Then lysis buffer #4 (2X) was added for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF phospho-PLCg2 (Tyr1217) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

Using the HTRF phospho-PLCg2 (Tyr1217) assay, 4,000 cells/well were enough to detect a significant signal, while 8,000 cells were needed to obtain a minimal chemiluminescent signal using Western Blot. Therefore in these conditions, the HTRF phospho-PLCg2 assay was 2 times more sensitive than the Western Blot technique.

Comparison of HTRF phospho-PLCg2 (Tyr1217) kit with western blot

Simplified pathway of PLCg2 signaling

PLCg2 (Phospholipase C gamma 2) is a cytoplasmic tyrosine kinase which plays a crucial role in signal transduction, especially in immune cells such as B cells and microglia (brain-resident macrophages).

Upon the stimulation of B cells or TREM2/DAP12 receptor complexes, PLCg2 interacts with SYK, BTK, and BLNK (also called SLP-65), leading to the activation by phosphorylation of its catalytic domain at Tyr1217. PLCg2 will then cleave the phospholipid phosphatidylinositol 4,5-bisphosphate (PIP2) into diacyl glycerol (DAG) and inositol 1,4,5-trisphosphate (IP3). The IP3 will then increase calcium release and degradate it into IP1.

This will trigger the activation of multiple signaling pathways and cellular responses, including cell activation, proliferation, and survival.

PLCG2 signaling pathway

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

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

Insider Tips for successful sample treatment - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

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

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

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

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

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

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

On-demand webinar: Linking Neuroinflammation and Neurodegeneration

New insight into neuroinflammation research - 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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