MAP-tau quantification in cell lysates
This HTRF kit enables the cell-based detection of phosphorylated alpha-synuclein at Ser129 as a marker of neurodegenerative diseases.
The HTRF Phospho-alpha Synuclein (Ser129) cell-based assay kit is ideal for quantifying endogenous phospho-alpha Synuclein phosphorylated on Serine 129.
The alpha Synuclein protein is involved in the pathogenesis of synucleinopathies, a group of neurodegenerative diseases that includes Parkinson’s disease (PD), dementia with Lewy bodies (DLB), diffuse Lewy body disease (DLBD), and multiple system atrophy (MSA).
The Phospho-alpha Synuclein (Ser129) assay measures Synuclein when phosphorylated at Ser129. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer.
The Phospho-Synuclein (Ser129) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody was selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving both the 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.
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 Phospho-alpha Synuclein (Ser129) HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.
Detection of Phospho-alpha Synuclein (Ser129) 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.
HEK293 cells were transfected with a plasmid encoding alpha-Synuclein prior to seeding treatment with non phosphorylated fibrils. After cell lysis, different dilutions of lysate were analyzed by HTRF.
Phosphorylation of alpha synuclein was detected in human brain homogenates from patients with Lewy Body Diseases.
The alpha Synuclein phosphorylation was detected on primary neurons obtained from WT mouse .
Inducible recombinant mouse N2A cells treated with non phosphorylated fibrils displayed high phosphorylation levels of alpha Synuclein.
Alpha-Synuclein is a prominent component of intracellular fibrillary aggregates in the brains of patients suffering from synucleinopathies. This protein represents a key molecular hallmark for Parkinson’s disease, dementia with Lewy bodies, or multiple system atrophy. The extensive phosphorylation of alpha synuclein on Ser129 is also associated with a pathological event.
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