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Phospho-Phospholamban (Thr17) & total Phospholamban Cellular Assay Kits   Phospho- and total Phospholamban assays for monitoring CaMKII activation Cisbio's cell-based homogeneous HTRF® phospho-Phospholamban (Thr17) immunoassay enables the quantitative detection of Phospholamban (PLN) phosphorylated on Threonine 17. The HTRF® total PLN assay kit is designed for the quantitative detection of total PLN, phosphorylated and unphosphorylated, to normalize the phosphorylation status of PLN with respect to its steady-state level in cells. 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. The increase in intracellular Ca2+ causes the activation of CaMKII. CaMKII in turn phosphorylates PLN at the Thr17 residue. Dephosphorylated PLN inhibits SERCA2a, whereas phosphorylation of PLN at either the Ser16 site by PKA or the Thr17 site by CaMKII reverses this SERCA2a inhibition. The PLN phosphorylation state regulates the activity of this Ca2+ pump. Amenable to low-volume formats, use of the HTRF® phospho- PLN (Thr17) assay ranges from basic research to high throughput drug screening.    Features Validated on Spontaneously hypertensive rat (SHR) heart homogenate Low-volume samples required Truly homogeneous, simple protocol with no wash steps More convenient and faster than ELISA or Western Blot   Applications Functional screening for Cardiac muscle studies Readout for CamKII Total Phospholamban for normalization     Assay Principle HTRF® - the homogeneous cell-based sandwich immunoassay  Cisbio Bioassay’s phospho- and total Phospholamban (PLN) assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-VASP antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind with PLN, and the proximity of donor and acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total PLN can be quantitatively detected using the HTRF phospho-Phospholamban (Thr17) and total Phospholamban cellular assay kit reagents, and results obtained with most TR-FRET multimode plate readers.     A simpler, more flexible assay protocol - adapted to your applications Two-plate assay protocol For added flexibility, the assay can be run under a two-plate assay protocol, where cells are first plated and treated in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384-well sv assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.        Product Performance  Validation of the HTRF phospho- and total phospholamban assays on Heart homogenate Spontaneously hypertensive (SHR) heart and normal heart samples were homogenized using Cisbio lysis buffer and supernatant was collected after centrifugation. 16µL of lysate were transferred into a 384-well sv white microplate and 4 µL of the HTRF phosphor-Phospholamban (Thr17) or total Phospholamban detection reagents were added. The HTRF signal was recorded after an overnight incubation at room temperature.     

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