This HTRF kit detects cellular LRKK2 modulation in the study of Parkinson's Disease It can be used as a normalization assay with the phospho-LRRK2 kit.

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  • Low sample consumption Low sample consumption
  • No-wash No-wash
  • Validated on PBMC Validated on PBMC

This HTRF kit detects cellular LRKK2 modulation in the study of Parkinson's Disease It can be used as a normalization assay with the phospho-LRRK2 kit.

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Overview

This assay is designed to monitor the modulation of LRKK2 protein levels and for use as a normalization assay with the phospho-LRRK2 kits. LRRK2 is a significant target in neuroscience research involving Parkinson’s disease (PD) and neurodegenerative disorders in general.

Benefits

  • VALIDATED ON PATIENT PBMC'S
  • SPECIFICITY

Total LRRK2 assay principle

The total LRRK2 assay quantifies the expression level of LRRK2 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The total LRRK2 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 LRRK2 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 LRRK2 assay principle

Total LRRK2 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 LRRK2 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-LRRK2 2-plate assay protocol

Total LRRK2 1-plate assay protocol

Detection of total LRRK2 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-LRRK2 1-plate assay protocol

HTRF total LRRK2 assay compared to Western Blot

Murine NIH-3T3 cells were cultured for 2 days until reaching 100% confluency. Following cell lysis, soluble fractions were then collected after a 10 min centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were dispensed and analyzed side-by-side by WB and by HTRF. The HTRF total assay is 2-fold more sensitive than the Western Blot: only 10,000 cells are sufficient for minimal signal detection when using the HTRF total LRRK2 assay while 20,000 cells are needed for a Western Blot.
HTRF total LRRK2 assay compared to Western Blot
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