HTRF Total RAB10 assay principle

The Total-RAB10 assay quantifies the expression level of RAB10 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Total-RAB10 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 RAB10 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-RAB10 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 Total-RAB10 HTRF detection reagents.

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

Total-RAB10 1-plate assay protocol

Detection of total RAB10 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 RAB10 detection on human and mouse 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 lysed with supplemented lysis buffer, and 4 µL of lysate (supplemented with 8 µL of lysis buffer) were transferred into a 384-well low volume white microplate before the addition of 2 µL of HTRF Phospho & Total Activation reagent A, 2 µL of HTRF Phospho & Total Activation reagent B, and then 4 µL of the HTRF Total RAB10 detection reagents. The HTRF signal was recorded after an overnight incubation.

The HTRF Total RAB10 assay efficiently detected RAB10 in various cellular models expressing different levels of the protein.

HTRF total RAB10 compared to Western-Blot

A549 cells were cultured in a T175 flask in complete culture medium at 37°C, 5% CO2. After a 48h incubation, the cells were stimulated with chloroquine 500 µM 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 12 µL of each dilution were transferred into a low volume white microplate before the addition of 2 µL of HTRF Phospho & Total Activation reagent A, 2 µL of HTRF Phospho & Total Activation reagent B, and 4 µL of HTRF Total-RAB10 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 32-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.