The total SMAD2 kit detects cellular SMAD2, and can be used as a normalization assay with our phospho-SMAD2 kit to enable optimal investigation of TGF-ß signaling.

See more
  • All inclusive kit All inclusive kit
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • Low sample consumption Low sample consumption

The total SMAD2 kit detects cellular SMAD2, and can be used as a normalization assay with our phospho-SMAD2 kit to enable optimal investigation of TGF-ß signaling.

In stock


The total SMAD2 cellular assay kit is designed to monitor the expression level of SMAD2, whether phosphorylated or unphosphorylated.

It is compatible with our Phospho-SMAD2 kit, and enables the analysis of phosphorylated and total proteins from a single sample for a better readout of TGF-ß signaling activity.



Total-SMAD2 assay principle

The Total-SMAD2 assay quantifies the expression level of SMAD2 in a cell lysate. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Total-SMAD2 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 SMAD2 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-SMAD2 assay principle

Total-SMAD2 2-plate assay protocol

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 Total-SMAD2 HTRF detection reagents.

This protocol enables the cells' viability and confluence to be monitored.

Total-SMAD3 2-plate assay protocol

Total-SMAD2 1-plate assay protocol

Detection of total SMAD2 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.

Total-SMAD3 1-plate assay protocol

Validation on the mouse cell lines C2C12 and human cell line HeLa

​HeLa and C2C12 cell lines were plated (100,000 cells/well) and cultured overnight in complete culture medium, 37°C - 5% CO2. The day after, medium was removed and the cells were incubated for 4h at 37°C - 5% CO2 with reduced culture medium (0.5% FBS). After incubation, the cells were treated with increasing concentrations of TGF-ß1 for 30 minutes at 37°C - 5% CO2. After medium removal, the cells were then lysed with 50 µL of supplemented lysis buffer #1 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 HTRF phospho-SMAD2 or total SMAD2 detection antibodies.

The HTRF signal was recorded after an overnight incubation. In both lines, TGF-ß1 promoted the activation of SMAD2 by phosphorylation on Ser465/467, whereas the expression level of the protein remained fairly stable.

Validation on of total SMAD2 kit on mouse C2C12 and human HeLa cell lines

TGF-ß signaling pathway

​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 to the nucleus. There, it 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.

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

Analyse of PI3K/AKT/mTor translational control pathway - Application Notes

Open R&D: Sanofi Access Platform

In collaboration with Sanofi - Scientific Presentations

Cisbio lysis buffer compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein Assays - Flyers

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Save time and money

Switch to HTRF assays - Flyers

Species compatibility

Cell Signaling: Biomarkers, Phospho- & total-protein assays - Flyers

HTRF cell-based phospho-protein data normalization

Valuable guidelines for efficiently analyzing and interpreting results - Application Notes

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

Increased flexibility of phospho-assays - Application Notes

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

Insider Tips for successful sample treatment - Technical Notes

HTRF Alpha-tubulin Housekeeping kit

Properly interpret your compound effect - Application Notes

HTRF assays facilitate investigation of the TGFB/SMAD/a-SMA signaling axis in liver fibrosis

Benefits of using HTRF assays to characterize liver fibrosis - Technical Notes

Optimize your HTRF cell signaling assays on tissues

HTRF and WB compatible guidelines - Technical Notes

Key guidelines to successful cell signaling experiments

Mastering the art of cell signaling assays optimization - Guides

HTRF phospho-assays reveal subtle drug-induced effects

Detailed protocol and direct comparison with WB - Posters

Webinar: NASH - Fibrosis Research and Drug Discovery

Featuring a panel of experts - Videos

How to run a cell based phospho HTRF assay

What to expect at the bench - Videos

Unleash the potential of your phosphorylation research with HTRF

Unmatched ease of use, sensitivity and specificity assays - Videos

Cisbio Product Catalog 2019

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

Product Insert SMAD2 total kit / 63ADK103PEG-63ADK103PEH

63ADK103PEG-63ADK103PEH - Product Insert

How HTRF compares to Western Blot and ELISA

Get the brochure about technology comparison. - Brochures

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

HTRF® cell signaling platform combined with iCell® Hepatocytes

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

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

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

Tumor xenograft analysis: HTRF versus Western blot - Application Notes

Side-by-side comparison of HTRF, Western Blot, ELISA and AlphaScreen® SureFire®

Do all cell-based kinase assays perform similarly? - Posters

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