Revvity, 64RABT73PEG, HTRF Human and Mouse Phospho-RAB10 (Thr73) Detection Kit, 500 Assay Points

Overview
RAS-related protein Rab10 (RAB10) belongs to the RAS superfamily of small GTPases. RAB10 is a key regulator of intracellular membrane trafficking by orchestrating the biogenesis, transport, tethering, and fusion of membrane-bound organelles and vesicles. LRRK2-mediated phosphorylation may cause deficits in endolysosomal trafficking pathways modulated by RAB10, contributing to the onset of neurodegenerative disease. RAB10 is associated with Alzheimer's Disease and Parkinson's Disease.
HTRF assays offer many advantages over other technologies:

Homogeneous add-and-read format
No wash steps
Low background
Straightforward miniaturization from 96- or 384-well microplates to high density assay formats such as 384-well low volume and 1536-well plates
Stable signal, providing flexibility in time of readout or size of assays
How it works
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. Application-Cell Signaling
Brand-HTRF
Detection Modality-HTRF
Lysis Buffer Compatibility-Lysis Buffer 3
Molecular Modification-Phosphorylation
Product Group-Kit
Sample Volume-16 µL
Shipping Conditions-Shipped in Dry Ice
Target Class-Phosphoproteins
Target Species-Human Mouse
Technology-TR-FRET
Unit Size-500 assay points