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HTRF Human and Mouse Phospho-RAB10 Thr73 Detection Kit

HTRF Human and Mouse Phospho-RAB10 Thr73 Detection Kit HTRF®

This HTRF kit enables the cell-based quantitative detection of RAB10 phosphorylation at Thr73, which is a key regulator of intracellular vesicular transport.

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All inclusive kit
Low sample consumption
No-wash
High sensitivity

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This HTRF kit enables the cell-based quantitative detection of RAB10 phosphorylation at Thr73, which is a key regulator of intracellular vesicular transport.

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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.

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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

Principle of the HTRF Rab10 phospho Thr73 assay

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.

2-plate protocol of the HTRF RAB10 phospho Thr73 assay

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.

1-plate protocol of the HTRF RAB10 Phospho Thr73 assay

Inhibition of Phospho RAB10-Thr73 on A549 and Raw264.7 cell lines

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.

Inhibition of phospho Rab10 Thr73 on A549 cell line

Inhibition of phospho Rab10 on Raw264.7 cell lines

Phospho RAB10-Thr73 down-regulation by siRNA

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.

RAB10 phospho Thr73 down regulation by siRNA

GAPDH RAB10 phospho Thr73 down regulation by siRNA

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.

Rab10 phospho Thr73 detection on various cell lines

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.

Comparison between HTRF and WB sensitivity on RAB10 phospho Thr73

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.

HTRF cellular phospho-protein assays

physiologically relevant results fo fast flowing research - Flyers

Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences

Insider Tips for successful sample treatment - Notes techniques

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Notes techniques

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Notes techniques

HTRF® phospho-protein platform facilitates the dissection of signaling pathways involved in insulin resistance and metabolic disorders

Multi-tissue cellular modeling and anlysis of insulin signaling - Posters

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - Posters

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Implement HTRF phospho-protein assays at every step of the drug discovery process, from in vitro to in vivo models

A single technology for 2D cells, 3D cells, and xenograft models - Posters

Simplify complex pathway dissection by combining the power of HTRF® cellular phospho-assays and the flexibility of the CyBi®-FeliX liquid handling system

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Universal HTRF® phospho-protein platform: from 2D, 3D, primary cells to patient derived tumor cells

Analysis of a large panel of diverse biological samples and cellular models - Posters

From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery

One technology across all samples - Notes d'application

HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts

Tumor xenograft analysis: HTRF versus Western blot - Notes d'application

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Notes d'application

HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers

Increased flexibility of phospho-assays - Notes d'application

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Notes d'application

Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

Analyse of PI3K/AKT/mTor translational control pathway - Notes d'application

How to run a cell based phospho HTRF assay

What to expect at the bench - Vidéos

Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening

In collaboration with Bayer - Présentations scientifiaues

Unleash the potential of your phosphorylation research with HTRF

A fun video introducing you to phosphorylation assays with HTRF - Vidéos

How to run a cell based phospho HTRF assay

3' video to set up your Phospho assay - Vidéos

Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection

Seeding and lysing recommendations for a number of cell culture vessels. - Notes techniques

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Notes d'application

Assessment of drug efficacy and toxicity by combining innovative technologies

Combination of AlphaLISA®, HTRF®, or AlphaLISA® SureFire® Ultra™ immunoassays with the ATPlite™ 1step cell viability assay - Notes d'application

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

Manual RAB10 p-T73 Kit / 64RABT73PEG-64RABT73PEH

64RABT73PEG-64RABT73PEH - Protocoles

Safety Data Sheet (DEU) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (ELL) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (FRA-FR) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (ITA) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (SPA) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (ENG-GB) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (ENG-US) RAB10 p-T73 Kit / 64RABT73PEG

64RABT73PEG - Fiches de sécurité

Safety Data Sheet (DEU) RAB10 p-T73 Kit / 64RABT73PEH