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.

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-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 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.