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HTRF Human Total Phospholamban Detection Kit, 10,000 Assay Points
HTRF Human Total Phospholamban Detection Kit, 10,000 Assay Points
generic HTRF total primary image
| Feature | Specification |
|---|---|
| Application | 細胞シグナル伝達 |
| Sample Volume | 16 µL |
Product variants
Unit Size: 500 assay points
Part #:
63ADK075PEG
Unit Size: 10,000 assay points
Part #:
63ADK075PEH
Unit Size: 50,000 assay points
Part #:
63ADK075PEY
For research use only. Not for use in diagnostic procedures. All products to be used in accordance with applicable laws and regulations including without limitation, consumption, and disposal requirements under European REACH regulations (EC 1907/2006).
HTRF Human Total Phospholamban Detection Kit, 10,000 Assay Points
generic HTRF total primary image
HTRF Human Total Phospholamban Detection Kit, 10,000 Assay Points
Product information
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.
How it works
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 proteins expression under a no-wash assay format.
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 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.
Assay validation
Total phospholamban assay on heart homogenate
Spontaneously hypertensive (SHR) heart and normal heart samples were homogenized using Revvity 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.
HTRF Total phospholamban assay compared to western blotnd HTRF
Spontaneously hypertensive (SHR) heart from rats were homogenized using Revvity 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.
Simplified pathway
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.
Specifications
| Application |
Cell Signaling
|
|---|---|
| Brand |
HTRF
|
| Detection Modality |
HTRF
|
| Lysis Buffer Compatibility |
Lysis Buffer 1
Lysis Buffer 3
Lysis Buffer 4
Lysis Buffer 5
|
| Molecular Modification |
Total
|
| Product Group |
Kit
|
| Sample Volume |
16 µL
|
| Shipping Conditions |
Shipped in Dry Ice
|
| Target Class |
Phosphoproteins
|
| Target Species |
Human
|
| Technology |
TR-FRET
|
| Therapeutic Area |
Cardiovascular
|
| Unit Size |
10,000 assay points
|
Video gallery
HTRF Human Total Phospholamban Detection Kit, 10,000 Assay Points
HTRF Human Total Phospholamban Detection Kit, 10,000 Assay Points
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Guide
HTRF solutions, guide to major applications
This guide provides you an overview of HTRF applications in several therapeutic areas.
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