Phospho-Phospholamban (Thr17) cellular kit

The phospho-Phospholamban (Thr17) kit enables the cell-based quantitative detection of phosphorylated Phospholamban at Thr17 (CaMKII activation).

Ready-to-use
High sensitivity
Low sample consumption
No-wash

The phospho-Phospholamban (Thr17) kit enables the cell-based quantitative detection of phosphorylated Phospholamban at Thr17 (CaMKII activation).

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Overview

This cell-based assay enables the detection of Phosphorylated Phospholamban (PLN) at Threonine 17. The phospho assay along with the total PLN assay are ideal for monitoring the activation of CaMKII. The increase in intracellular Ca2+ causes the activation of CaMKII, which in turn phosphorylates PLN at the Thr17 residue. Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this SERCA2a inhibition. The PLN phosphorylation state regulates the activity of this Ca2+ pump.

Benefits

SPECIFICITY
PRECISION
DATA NORMALIZATION

Phospho-Phospholamban (Thr17) assay principle

The Phospho-Phospholamban (Thr17) assay measures Phospholamban when phosphorylated at Thr17. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-Phospholamban (Thr17) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent 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 proteins phosphorylation state under a no-wash assay format.

Phospho-Phospholamban (Thr17) 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 Phospho-Phospholamban (Thr17) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospho-Phospholamban (Thr17)1-plate assay protocol

Detection of Phosphorylated Phospholamban (Thr17)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.

Phospho-Phospholamban (Thr17) 1-plate protocol

Phospho-phospholamban assay on heart homogenate

Spontaneously hypertensive (SHR) heart and normal heart samples were homogenized using Cisbio lysis buffer and supernatant was collected after centrifugation. 16 µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phospho-Phospholamban (Thr17) detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

Phospho-phospholamban (Thr17) assays on Heart homogenate

Simplified pathway for PLN

Phospholamban (PLN) plays a crucial role in heart failure through its control of cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA2a). This protein has a Ca2+ pump included in the SR membrane, and once activated, Ca2+ goes inside SR. Insufficient SERCA2a activity is a hallmark of heart failure. Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses the inhibition of SERCA2a. The PLN phosphorylation state regulates the activity of this Ca2+ pump. Relaxation is driven by dephosphorylation of PLN, and contraction by the phosphorylation status.

This small protein, is present in cardiac, smooth, and slow-twitch skeletal muscles. However, its regulatory effects have mainly been studied in cardiac muscle. The activation process is not well known, but 2 main pathways are described ending in 2 different phosphaorylations: on residue Serine 16, or on Threonine 17. Binding of a β-agonist to its receptor activates G protein, which enhances adenylate cyclase (AC) activity. AC catalyzes cAMP formation, which activates PKA. PKA phosphorylates the L-type Ca2+ channel, increasing the Ca2+ influx, and phosphorylates PP1 and the Ser16 residue of PLN. An increase in intracellular Ca2+ causes the activation of CaMKII. This autophosphorylation state is also controlled by PP1. CaMKII in turn phosphorylates PLN at the Thr17 residue.

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

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Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio 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

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Do all cell-based kinase assays perform similarly? - Posters

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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

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One technology across all samples - Application Notes

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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

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Optimize your HTRF cell signaling assays on tissues

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Key guidelines to successful cell signaling experiments

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Detailed protocol and direct comparison with WB - Posters

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

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How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

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Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening

In collaboration with Bayer - Scientific Presentations

Product Insert PLamban P-T17 Kit / 63ADK074PEG

63ADK074PEG - Product Insert

Product Insert PLamban P-T17 Kit / 63ADK074PEH

63ADK074PEH - Product Insert

Cisbio Product Catalog 2019

All your HTRF assays in one document! - Catalog

A guide to Homogeneous Time Resolved Fluorescence

General principles of HTRF - Guides

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Get the brochure about technology comparison. - Brochures

HTRF® cell signaling platform combined with iCell® Hepatocytes

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Unleash the potential of your phosphorylation research with HTRF

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

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3' video to set up your Phospho assay - Videos

Safety Data Sheet (DEU-) PLamban P-T17 Kit / 63ADK074PEG