Total alpha Synuclein assay principle

The Total-alpha Synuclein assay quantifies the expression level of Synuclein in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer.

The Total-alpha Synuclein 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 Synuclein 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 protein’s expression under a no-wash assay format.

Total Synuclein 2-plate assay protocol

The 2 plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates into a 384-well low volume detection plate before adding Total-Synuclein HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Total alpha Synuclein 1-plate assay protocol

Detection of total alpha Synuclein 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.

Alpha synuclein level, quantified by HTRF, is more abundant in Hela cells compared to SH-SY5Y

Hela and SH-SY5Y cells were seeded and incubated until confluence at 37°C, 5% CO2.

After cell lysis, the lysates were serially diluted. The total protein content was then quantified using BCA reagent, while alpha Synuclein was quantified with the HTRF Total alpha Synuclein cellular kit.

Specifically quantification of the alpha Synuclein, not the beta Synuclein

HTRF total alpha Synuclein does not detect the beta-Synuclein protein.