Revvity, 64PLN16PEH, HTRF Human Phospho-Phospholamban (Ser16) Detection Kit, 10,000 Assay Points

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.
How it works
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.

 

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.

 

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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 1 Lysis Buffer 2 Lysis Buffer 3 Lysis Buffer 4 Lysis Buffer 5
Molecular Modification-Phosphorylation
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