HTRF Human Total Phospholamban Detection Kit, 500 Assay Points

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.

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.