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Phospho-Phospholamban (Ser16) cellular kit HTRF®

This HTRF kit enables the cell-based quantitative detection of phosphorylated Phospholamban at Ser16.

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

This HTRF kit enables the cell-based quantitative detection of phosphorylated Phospholamban at Ser16.

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Overview

This cell-based assay enables the detection of Phosphorylated Phospholamban (PLN) at Serine 16. The phospho assay along with the total PLN assay are ideal for monitoring the activation of PKA in response GPCR stimulation, such as the beta2 Adrenergic receptor. PKA phosphorylates PLN at the Ser 16 residue.

In an unphosphorylated state, PLN inhibits SERCA2a which is the Ca2+ pump controling the contractility of the cardiac muscle. PLN phosphorylation induced by PKA and CaMKII, on Ser16 and Thr17 sites respectively, abrogates  SERCA2a inhibition. The PLN phosphorylation state regulates the activity of this Ca2+ pump in the heart.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Phospho-Phospholamban (Ser16) assay principle

The Phospho-Phospholamban (Ser16) assay measures Phospholamban when phosphorylated at Ser16. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Phospho-Phospholamban (Ser16) 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 from 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.

Phospholamban phospho-S16 assay principle

Phospho-Phospholamban (Ser16) 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 (Ser16) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.

Phospholamban phospho-S16 2-plate assay protocol

Phospho-Phospholamban (Thr17)1-plate assay protocol

Detection of Phosphorylated Phospholamban (Ser16)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.

Phospholamban phospho-S16 1-plate assay protocol

Phospho Ser16-phospholamban assay on heart homogenate

Spontaneously hypertensive (SHR) heart and wistar Kyoto (WKY) heart samples were homogenized using Cisbio lysis buffer, and supernatant was collected after centrifugation. Total protein were quantified and a side by side dilution was done in lysis buffer. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-Phospholamban (Ser16) detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.

Phospho Ser16-phospholamban assay on heart homogenate

Phospho-phospholamaban comparison on hypertensive rat heart lysates

Spontaneously hypertensive (SHR) heart and wistar Kyoto (WKY) heart samples were homogenized using Cisbio lysis buffer, and supernatant was collected after centrifugation. On a fixed total protein concentration (16.25 µg/mL), a side by side comparison was done between the HTRF phospho Trh17 PLN kit and the phospho-Ser16 PLN kit. In order to normalize the detection, the total phospholamban was also detected. 16 µL of lysate were transferred into a 384-well sv white microplate, and 4 µL of the HTRF phospho-Phospholamban (Ser16) detection reagents or the phospho-Phospholamban (Thr17) detection reagents or total phospholamban were added. The HTRF signal was recorded after an overnight incubation at room temperature. The data is presented after a normalization on the total expression level.

**** t-test analysis P value <0.0001

HTRF Phospho Ser16-PLN and phosphoThr17-PLN assay on hypertensive and normal rat heart lysates

HTRF phospho Ser16-PLN assay compared to Western Blot

SHR heart homogenates were collected after centrifuging for 10 minutes. Equal amounts of total were used for a side by side comparison of WB and HTRF. 110 µg/mL of total protein were used as the highest concentration of a serial dilution carried out. HTRF phospho-PLN assay shows better sensitivity compared to WB, as 0.49µg/mL of total protein was sufficient to detect the phosphorylation, much lower than 13.3 µg/mL for WB.

Phospho-phospholamban detection on WB and HTRF

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 for PLN

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

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

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

HTRF Product Catalog 2020 July update

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

Product Insert PLamban P-S16 Kit / 64PLN16PEG-64PLN16PEH

64PLN16PEG-64PLN16PEH - Product Insert

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