Description
Alpha-Synuclein (α-Syn) is a small, intrinsically disordered protein predominantly expressed in neurons, particularly in presynaptic terminals. It plays a crucial role in synaptic function, vesicle trafficking, neurotransmitter release and synaptic plasticity. In its normal state, α-Syn exists as a soluble monomer, but it can misfold and aggregate into oligomers, fibrils, and Lewy bodies. These aggregates are a hallmark of synucleinopathies, including Parkinson's disease (PD), Dementia with Lewy Bodies (DLB), and Multiple System Atrophy (MSA). Misfolded α-Syn disrupts cellular homeostasis, impairs synaptic function, and propagates toxic fibrils between neurons in a prion-like manner, leading to neurodegeneration. Therapeutic strategies aim to reduce α-Syn aggregation, enhance its degradation, and prevent its pathological spread.
The AlphaLISA SureFire Ultra Human and Mouse Phospho-α-Synuclein (Ser129) Aggregate Detection Kit is a sandwich immunoassay for the quantitative detection of phospho-α-Synuclein (Ser129) aggregate in cellular lysates, using Alpha Technology.
Formats:
The HV (high volume) kit contains reagents to run 100 wells in 96-well format, using a 60 μL reaction volume.
The 500-point kit contains enough reagents to run 500 wells in 384-well format, using a 20 μL reaction volume.
The 10,000-point kit contains enough reagents to run 10,000 wells in 384-well format, using a 20 μL reaction volume.
The 50,000-point kit contains enough reagents to run 50,000 wells in 384-well format, using a 20 μL reaction volume.
AlphaLISA SureFire Ultra kits are compatible with:
Cell and tissue lysates
Antibody modulators
Biotherapeutic antibodies
AlphaLISA SureFire Ultra kits can be used for:
Cellular kinase assays
Receptor activation studies
High-throughput screening for preclinical studies
Phospho Aggregate-AlphaLISA SureFire Ultra assay principle
The Phospho Aggregate-AlphaLISA SureFire Ultra assay measures the level of phosphorylated aggregates of a target protein in a biological sample (e.g. cell lysate).
This assay uses a single phospho-specific antibody clone tagged with both CaptSure™ peptide and biotin, that recognizes an exposed phospho-epitope on the aggregated target protein. AlphaLISA assays require two bead types: Acceptor and Donor Beads. Acceptor Beads are coated with a proprietary CaptSure agent to specifically immobilize the antibody labeled with the CaptSure tag. Donor Beads are coated with Streptavidin to capture the same antibody which is biotinylated. In the presence of phosphorylated aggregate protein, the Donor and Acceptor Beads are brought into close proximity whereby the singlet oxygen transfers energy to excite the Acceptor Bead, allowing for the generation of a luminescent Alpha signal. The amount of light emission is directly proportional to the quantity of phosphorylated aggregate protein present in the sample.
Phospho Aggregate-AlphaLISA SureFire Ultra two-plate assay protocol
The two-plate protocol involves culturing and treating the cells in a 96-well plate before lysis, then transferring lysates into a 384-well OptiPlate™ plate before the addition of Phospho Aggregate-AlphaLISA SureFire Ultra detection reagents. This protocol enables cell viability and confluence to be monitored. In addition, lysates from a single well can be used to measure multiple targets.
Phospho Aggregate-AlphaLISA SureFire Ultra one-plate assay protocol
Detection of aggregated target protein with AlphaLISA SureFire Ultra reagents can be performed in a single plate used for culturing, treatment, and lysis. No washing steps are required. This HTS designed protocol allows for miniaturization while maintaining robust AlphaLISA SureFire Ultra quality.
Selectivity of α-Synuclein Phospho (Ser129) Aggregate assay
Selectivity of the α-Synuclein Phospho (Ser129) Aggregate assay was assessed by assaying aggregated and monomeric proteins. Phosphorylated α-Synuclein aggregate (Abcam ab218819) and phosphorylated α-Synuclein monomer were prepared at approximately 250 ng/mL in Lysis Buffer and evaluated using the Phospho (Ser129) Aggregate AlphaLISA SureFire Ultra assay.
For the detection step, 10 µL of protein was transferred into a 384-well white OptiPlate, followed by 5 µL of Acceptor mix and incubated for 1 hour at room temperature. Finally, 5 µL of Donor mix was then added to each well and incubated for 1 hour at RT in the dark. The plate was read on an Envision using standard AlphaLISA settings.
α-Synuclein Phospho (Ser129) Aggregate assay recognizes phosphorylated α-Synuclein protein only when it is aggregated.
