Alpha-Synuclein aggregation Kit
Alpha-Synuclein aggregation detection in cell and brain extracts
This HTRF kit enables the cell-based quantitative detection of RAB10 phosphorylation at Thr73, which is a key regulator of intracellular vesicular transport.
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.
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
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.
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.
A549 and Raw264.7 cells were seeded in a 96-well culture-treated plate under 200,000 cells / well in complete culture medium, and incubated overnight at 37°C, 5% CO2. The cells were then treated with increasing concentrations of MLi-2 for 1H30 at 37°C, 5% CO2, followed by stimulation with chloroquine at 100 µM for 3 hours.
After cell lysis, 16 µL of lysate were transferred into a 384-well sv white microplate before the addition of 4 µL of HTRF Phospho (Thr73) detection reagents for the detection of the level of phosphorylated protein. In parallel, 4 µL of lysate (supplemented with 8 µL of lysis buffer) were also transferred before the addition of 2 µL of HTRF Phospho & Total Activation reagent A, 2 µL of HTRF Phospho & Total Activation reagent B, then 4 µL of Total RAB10 detection reagents for the detection of the Total RAB10 protein. The HTRF signal was recorded after an overnight incubation at room temperature.
As expected, the results obtained showed a dose-response inhibition of RAB10 Thr73 phosphorylation upon treatment with MLi-2, while the RAB10 expression level remained constant.
A549 cells were plated in 96-well plates (100,000 cells/well) and cultured for 24h. The cells were then transfected with siRNAs specific for RAB10 as well as with a negative control siRNA. After a 48h incubation, the cells were stimulated with 500 µM chloroquine for 5 hours and then lyzed with supplemented lysis buffer. 16 µL of lysate were transferred into a 384-well low volume white microplate before the addition of 4 µL of the HTRF Phospho RAB10 (Thr73) detection antibodies. An additional 4 µL of lysates (supplemented with 12 µL diluent #11) were also transferred into the microplate to monitor the GAPDH level using the GAPDH Housekeeping Cellular Kit (64GAPDHPET/G/H). HTRF signals for both kits were recorded after an overnight incubation.
Cell transfection with RAB10 siRNA led to a 64% signal decrease compared to the cells transfected with the negative siRNA. The level of GAPDH measured remained unchanged under all the conditions tested. The data demonstrate that the HTRF Phospho RAB10 (Thr73) kit is specific for the detection of the phosphorylated RAB10 protein.
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.
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.
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.
physiologically relevant results fo fast flowing research - Flyers
Insider Tips for successful sample treatment - Notes techniques
HTRF and WB compatible guidelines - Notes techniques
Protocol for tumor xenograft analysis with HTRF - Notes techniques
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
A solution for phospho-protein analysis in metabolic disorders - Posters
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
PI3K/AKT/mTor translational control pathway - Posters
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Notes d'application
Tumor xenograft analysis: HTRF versus Western blot - Notes d'application
Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application
Increased flexibility of phospho-assays - Notes d'application
Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application
In collaboration with Bayer - Présentations scientifiaues
A fun video introducing you to phosphorylation assays with HTRF - Vidéos
Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques
Learn how to reduce time and sample consumption - Notes d'application
Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Notes d'application
Mastering the art of cell signaling assays optimization - Guides
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Total alpha Synuclein cellular kit
Simple, all-in-one kit for robust detection of total alpha Synuclein