Alpha-SMA quantification in cell lysates
This HTRF cell-based assay enables the rapid, quantitative detection of SMAD2 phosphorylated at Serine 465/467, as a readout of TGF-ß signaling activity.
TGF-ß receptors directly activate SMAD2 by phosphorylation at Ser465/467, causing it to translocate to the nucleus and regulate gene expression involved in apoptosis, migration, and differentiation, as well as in immune/inflammatory responses and extracellular matrix remodeling.
The Phospho-SMAD2 (Ser465/4267) assay measures SMAD2 when phosphorylated at Ser465/4267. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.
The Phospho-SMAD2 (Ser465/4267) assay uses 2 labeled antibodies: one with a donor fluorophore, the other with an acceptor. The first antibody is 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 both 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 to a 384-well low volume detection plate before adding Phospho-SMAD2 (Ser465/467) HTRF detection reagents.
This protocol enables the cells' viability and confluence to be monitored.
Detection of Phosphorylated SMAD2 (Ser465/467) 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.
C2C12 cells were plated at various cellular densities in a 96-well plate. After an overnight incubation at 37°C, 5% CO2, a serial dilution of human TGFβ was added to the cells for 30 minutes at 37°C, 5% CO2. Stimulation medium was removed, and 50µL of lysis buffer was added to the cells. A lysis step was carried out, shaking gently for 30 minutes. 16µL of samples were transferred into a 384-well small volume plate, then 4µL of Phospho-SMAD2 HTRF detection reagents were added. Signals were recorded overnight.
Hela cells were selected for testing human compatibility, while NIH 3T3 and C2C12 cells were chosen for mouse compatibility. 100,000 cells of these different models were plated in 96-well plates. After an overnight incubation at 37°C, 5% CO2, a serial dilution of human TGFβ was added to the cells for 30 minutes at 37°C, 5% CO2. Stimulation medium was removed, and 50µL of lysis buffer was added to the cells. A lysis step was carried out, shaking gently for 30 minutes. 16µL of samples were transferred into a 384-well small volume plate, then 4µL of Phospho-SMAD2 HTRF detection reagents were added. Signals were recorded overnight.
The Phospho-SMAD2 HTRF assay was able to detect human and mouse versions of this protein under its phosphorylated status on Serine 465/467.
Mouse C2C12 cells were cultured to 80% confluency. After hTGFβ treatment, cells were lysed and soluble supernatants were collected via centrifugation. Serial dilutions of the cell lysate were performed and 16 µL of each dilution were transferred into a 384-well low volume white microplate before finally adding Phospho-SMAD2 HTRF cellular kit reagents. A side by side comparison showed the HTRF Phospho assay is at least 32-fold more sensitive than the Western Blot.
TGF-ß signaling is mediated by complexes of TßRI and TßRII, which activate intracellular SMAD3 and SMAD2 by phosphorylation. The binding of the TGF-ß ligand on TßRII triggers the recruitment of TßRI into the ligand-receptor complex. TßRII autophosphorylates, then transphosphorylates TßRI. Activated TßRI in turn phosphorylates SMAD2 on Ser465 and Ser467, enabling its oligomerization with SMAD4. This complex then translocates into the nucleus, and acts as a transcription factor with coactivators and corepressors to regulate the expression of multiple genes involved in cell growth, apoptosis, proliferation, migration, and differentiation, as well as in extracellular matrix remodeling and immune/inflammatory responses. Inhibitory SMAD6 and SMAD7 are involved in feedback inhibition of the pathway.
Analyse of PI3K/AKT/mTor translational control pathway - Application Notes
Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers
Physiologically relevant results fo fast flowing research - Flyers
Analysis of a large panel of diverse biological samples and cellular models - Posters
One technology across all samples - Application Notes
Tumor xenograft analysis: HTRF versus Western blot - Application Notes
Valuable guidelines for efficiently analyzing and interpreting results - Application Notes
Increased flexibility of phospho-assays - Application Notes
Multi-tissue cellular modeling and anlysis of insulin signaling - Posters
Insider Tips for successful sample treatment - Technical Notes
Benefits of using HTRF assays to characterize liver fibrosis - Technical Notes
HTRF and WB compatible guidelines - Technical Notes
Mastering the art of cell signaling assays optimization - Guides
Detailed protocol and direct comparison with WB - Posters
A single technology for 2D cells, 3D cells, and xenograft models - Posters
Protocol for tumor xenograft analysis with HTRF - Technical Notes
Unmatched ease of use, sensitivity and specificity assays - Videos
In collaboration with Bayer - Scientific Presentations
Useful overview of today’s NAFLD knowledge - Guides
Get the brochure about technology comparison. - Brochures
A solution for phospho-protein analysis in metabolic disorders - Posters
PI3K/AKT/mTor translational control pathway - Posters
A fun video introducing you to phosphorylation assays with HTRF - Videos
63ADK102PEG-63ADK102PEH - Product Insert
64SMAD2S5PEG-64SMAD2S5PEH - Product Insert
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