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Total Phospholamban cellular kit HTRF®

The Total-Phospholamban kit can be used as a normalization assay for the phospho-Phospholamban kit.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Low sample consumption Low sample consumption
  • No-wash No-wash
The Total-Phospholamban kit can be used as a normalization assay for the phospho-Phospholamban kit.
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Overview

Total Phospholamban (PLN) cellular assay kit serves as a normalization assay with our Phospho-PLN kits, it is designed for the quantitative detection of total PLN, phosphorylated and unphosphorylated. The buffers of the HTRF phospho- and total PLN assays are compatible, enabling an analysis of the phosphorylated and the total protein populations from one lysate sample.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-Phospholamban assay principle

The Total-Phospholamban assay quantifies the expression level of Phospholamban in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-Phospholamban 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 Phospholamban 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-Phospholamban assay principle

Total-Phospholamban 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-Phospholamban HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-Phospholamban 2-plate assay protocol

Total-Phospholamban 1-plate assay protocol

Detection of total Phospholamban 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-Phospholamban 1-plate protocol

Total 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 total Phospholamban detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.
Total phospholamban (Thr17) assays on Heart homogenate

HTRF Total phospholamban assay compared to western blotnd HTRF

Spontaneously hypertensive (SHR) heart from rats were homogenized using Cisbio lysis buffer and supernatant was collected after 10-minute centrifugation. An HTRF Total-PLN assay was performed following the package insert instructions and equal amounts of sample were used in a side by side comparison with WB.

The HTRF total-PLN assay showed an improved sensitivity compared to WB and was able to detect PLN at 1.5µg/mL whereas the WB detection threshold was 13 µg/mL.


Total 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

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

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 total Kit / 63ADK075PEG-63ADK075PEH

63ADK075PEG-63ADK075PEH - Product Insert

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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