HTRF phospho RAB10 assay principle

The Phospho-RAB10 (Thr73) assay measures RAB10 when phosphorylated at Threonine 73. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-RAB10 (Thr73) 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, and the second for its ability to recognize the protein independently of its phosphorylation state. Protein phosphorylation enables an immune-complex formation involving the two 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

HTRF Phospho-RAB10 Thr73 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 the addition of the phospho-RAB10 Thr 73 HTRF detection reagents.

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

HTRF Phospho-RAB10 Thr73 one-plate assay protocol

Detection of Phospho RAB10 Thr 73 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.

Detection of Phospho RAB10 (Thr73) on various cell lines

A549, 1321-N1 and Raw264.7 cells were seeded at 200,000 cells / well in a 96-well microplate. After a 24h incubation, the cells were stimulated or not with chloroquine 500 µM for 5 hours, then lysed with supplemented lysis buffer. Next,16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL HTRF Phospho RAB10 (Thr73) detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Phospho Rab10 (Thr73) assay efficiently detected phospho RAB10 (Thr73) in various cellular models stimulated or not with chloroquine.

HTRF Phospho RAB10-Thr73 compared to Western Blot

A549 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 72h incubation, the cells were stimulated with chloroquine for 5 hours and then lysed with 3 mL of supplemented lysis buffer #3 (1X) for 30 minutes at RT under gentle shaking.

Serial dilutions of the cell lysate were performed using supplemented lysis buffer, and 16 µL of each dilution were transferred into a low volume white microplate before the addition of 4 µL of HTRF Phospho-RAB10 (Thr73) detection reagents. Equal amounts of lysates were used for a side by side comparison between HTRF and Western Blot.

The side by side comparison of Western Blot and HTRF demonstrates that the HTRF assay is 8-fold more sensitive than the Western Blot, at least under these experimental conditions.

Simplified RAB10 signaling pathway

Leucine-rich repeat kinase 2 (LRRK2), the major causative gene of autosomal-dominant Parkinson's Disease, is a protein kinase that phosphorylates a subset of RAB GTPases including RAB10. RAB 29 mediates the recruitment of LRRK onto organelle membranes, and enhances LRRK2 enzymatic activity (monitored by Ser1292 autophosphorylation). LRRK2 induces phosphorylation of RAB10 (Thr73), which in turn regulates ciliogenesis, vesicle transport, membrane trafficking, and fusion.