Alpha-SMA kit
Alpha-SMA quantification in cell lysates
Total FAK cellular kit HTRF®
The Total FAK kit is designed to monitor the expression level of cellular FAK, and can be used as a normalization assay for the phospho-FAK kit.
-
All inclusive kit
-
High sensitivity
-
No-wash
-
Low sample consumption
Overview
The Total FAK cellular assay monitors total FAK and is used as a normalization assay with the phospho-FAK kit. This kit is compatible with the buffers from the phospho-FAK kit, so the same lysate can be used for analyses of both the phosphorylated and the total protein populations.
FAK, Focal Adhesion Kinase, is a cytoplasmic tyrosine kinase controlling cell adhesion, migration, proliferation, and survival. FAK is a central regulator of ECM/integrin and cadherin signaling, and plays a role in cell adhesion, cell junction, migration, survival, and mechanosensing. FAK can be activated by Growth Factor Receptors, Cytokine Receptors, and GPCRs. Increased FAK expression and phosphorylation is associated with increased tumor cell adhesion, migration, invasion in several cancers, and with fibroblast migration on fibronectin in tissue fibrosis.
Benefits
- SPECIFICITY
- PRECISION
- DATA NORMALIZATION
Total-FAK assay principle
The Total FAK assay quantifies the expression level of FAK in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer. The Total-FAK 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 FAK 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-FAK two-plate assay protocol
The two-plate protocol involves culturing cells in a 96-well plate before lysis, then transferring lysates to a 384-well low volume detection plate before the addition of Total-FAK HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-FAK one-plate assay protocol
Detection of Total FAK 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.
Accumulation of phosphorylated FAK (Tyr397) in human cancer cell lines
Human MCF7 or HeLa cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, cells were treated for 10 min in the presence or absence of Pervanadate. After medium removal, cells were lysed with 50 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-FAK (Tyr397) or Total-FAK detection reagents. The HTRF signal was recorded after an overnight incubation.
Treatment with pervanadate, a phosphatase inhibitor, lead to a significant increase in the phosphorylation of FAK on Tyrosine 397. The total amount of FAK protein remained unaffected by the pervanadate treatment.
HTRF phospho and Total FAK assays to monitor pharmacology of FAK inhibitors
NIH/3T3 mouse fibroblasts or human HeLa cells were plated in a 96-well plate (100,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, cells were treated with increasing concentrations of FAK inhibitors for 30 min. After medium removal, cells were then lysed with 50 µL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-FAK (Tyr397) or Total-FAK detection reagents. The HTRF signal was recorded after an overnight incubation.
Treatment with PF-562271 lead to a dose dependent decrease in FAK phosphorylation on Tyrosine 397, in both Human HeLa and Mouse NIH/3T3 cell lines. The total amount of FAK protein remained unaffected by the pervanadate treatment.
HTRF FAK assays as readouts to monitor growth factor receptor activation
HUVEC human endothelial cells were plated in a 24-well collagen I plate (400,000 cells/well) in complete culture medium and incubated at 37°C, 5% CO2. The day after, cells were incubated in serum-free conditions. The following day, cells were treated with increasing concentrations of VEGF for 5 min in serum-free conditions. After medium removal, cells were then lysed with 250 µL supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking, and 16 µL of lysate were transferred twice over into a low volume white microplate before the addition of 4 µL of the premixed HTRF phospho-FAK (Tyr397) or Total-FAK detection reagents. The HTRF signal was recorded after an overnight incubation.
HTRF phospho-FAK cellular assays compared to Western Blot
The mouse NIH/3T3 cell line was seeded in a T175 flask in complete culture medium, and incubated for 2 days at 37°C, 5% CO2, until 90% confluency was reached. Cells were then lysed with 3 mL of supplemented lysis buffer #3 for 30 minutes at RT under gentle shaking. Soluble supernatants were collected after a 10-minute centrifugation.
Serial dilutions of the cell lysate were performed in the 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-FAK detection reagents. Equal amounts of lysates were used to establish a side-by-side comparison between HTRF and Western Blot.
Using the HTRF® Total-FAK kit, just 2,500 cells/well were sufficient to detect a significant signal while 5,000 cells were needed for minimal ECL signal detection using Western Blot. The HTRF assay is therefore twice as sensitive as the Western Blot technique.
FAK simplified pathway
This multi-domain protein changes conformation upon activation by cell surface receptors, mainly by integrins, cytokine receptors (IL1βR, TNFαR), growth factor receptors (EGFR, VEGFR, PDGFR), and GPCRs (LPA, bombesin). The cytoplasmic inactive FAK monomer is recruited to the plasma membrane by activated receptors, that yield to a transient dimerization and subsequent activation by autophosphorylation on tyrosine 397. This is a docking site for Src family kinases, allowing phosphorylation of other residues. Fully activated FAK displays binding sites for FAK substrates including Grb2, PI3K, and cadherins. FAK serves as a central regulator of ECM/integrin signaling, is required in the regulation of assembly/disassembly of focal adhesions, and plays a role in cell adhesion, migration, survival, and mechanosensing. In cadherin signaling, the regulation of adherens junctions between endothelial/epithelial cells enables FAK to play a role in cell junction, vascular permeability, and angiogenesis. FAK can activate various substrates and regulate multiple signaling pathways, such as Rho GTPases/actin (cytoskeleton reorganization/cell migration), Cadherin/β-catenin (cell junctional stability/vascular permeability), PI3K/AKT (cell survival), MAPKs ERK & JNK (cell proliferation), and p53/MDM2 (anti-apoptosis).
HTRF phospho-assays reveal subtle drug-induced effects
Detailed protocol and direct comparison with WB - Posters
HTRF® cell signaling platform combined with iCell® Hepatocytes
A solution for phospho-protein analysis in metabolic disorders - Posters
Key guidelines to successful cell signaling experiments
Mastering the art of cell signaling assays optimization - Guides
Optimize your HTRF cell signaling assays on tissues
HTRF and WB compatible guidelines - Technical Notes
HTRF Alpha-tubulin Housekeeping kit
Properly interpret your compound effect - Application Notes
Best practices for analyzing brain samples with HTRF® phospho assays for neurosciences
Insider Tips for successful sample treatment - Technical Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
HTRF phospho-total lysis buffer: a universal alternative to RIPA lysis buffers
Increased flexibility of phospho-assays - Application Notes
HTRF cell-based phospho-protein data normalization
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
HTRF phospho assays reveal subtle drug induced effects in tumor-xenografts
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
From 2D, 3D cell cultures to xenografts: A smart HTRF platform to maximize anticancer drug discovery
One technology across all samples - Application Notes
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Best practices for analyzing tumor xenografts with HTRF phospho assays
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Cell-based kinase assays in HTS ? potential and limitations for primary and secondary screening
In collaboration with Bayer - Scientific Presentations
Unleash the potential of your phosphorylation research with HTRF
Unmatched ease of use, sensitivity and specificity assays - Videos
How to run a cell based phospho HTRF assay
What to expect at the bench - Videos
TCR signaling investigation with HTRF phospho assays
Study a pathway of interest in PBMC and T cells - Application Notes
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
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Investigating kinase activity in a cellular context
HTRF cellular assays - Scientific Presentations
PI3K/AKT/mTor translational control pathway - Posters
Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers
Biomarker and Cell Signaling Assays for Fibrosis and NASH
HTRF solutions for NASH - Flyers
HTRF cellular phospho-protein assays
Physiologically relevant results fo fast flowing research - Flyers
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
Open R&D: Sanofi Access Platform
In collaboration with Sanofi - Scientific Presentations
HTRF solutions for each cell type - Flyers
Product Insert FAK total Kit / 64NAKPEG-64NAKPEH
64NAKPEG-64NAKPEH - Product Insert
All your HTRF assays in one document! - Catalog
A guide to Homogeneous Time Resolved Fluorescence
General principles of HTRF - Guides
A collection of the most important NAFLD pathways
Useful overview of today’s NAFLD knowledge - Guides
How HTRF compares to Western Blot and ELISA
Get the brochure about technology comparison. - Brochures
Unleash the potential of your phosphorylation research with HTRF
A fun video introducing you to phosphorylation assays with HTRF - Videos
How to run a cell based phospho HTRF assay
3' video to set up your Phospho assay - Videos
Guidelines for Cell Culture and Lysis in Different Formats Prior to HTRF Detection
Seeding and lysing recommendations for a number of cell culture vessels. - Technical Notes
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 - Application Notes
Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)
Learn how to reduce time and sample consumption - Application Notes
Safety Data Sheet (DEU) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (ELL) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (FRA-FR) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (ITA) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (SPA) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (ENG-GB) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (ENG-US) FAK total Kit / 64NAKPEG
64NAKPEG - Safety Data Sheet
Safety Data Sheet (DEU) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (ELL) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (FRA-FR) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (ITA) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (SPA) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (ENG-GB) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (ENG-US) FAK total Kit / 64NAKPEH
64NAKPEH - Safety Data Sheet
Safety Data Sheet (DEU) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Safety Data Sheet (ELL) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Safety Data Sheet (FRA-FR) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Safety Data Sheet (ITA) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Safety Data Sheet (SPA) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Safety Data Sheet (ENG-GB) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Safety Data Sheet (ENG-US) FAK total Kit / 64NAKPEY
64NAKPEY - Safety Data Sheet
Plate Reader Requirement
Choosing the right microplate reader ensures you’ll get an optimal readout. Discover our high performance reader, or verify if your lab equipment is going to be compatible with this detection technology.
Let's find your reader
You might be interested in