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Total-SMAD2 cellular kit HTRF®

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.
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  • 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.
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Overview

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.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

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

Human TGFβ stimulation on C2C12 cells

C2C12 cells were plated at 50,000 cells per well 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. In parallel, 16µl of samples were dispensed into other wells, then 4µL of Total-SMAD2 HTRF detection reagents were added. Signals were recorded overnight.

Human TGFβ stimulation on C2C12 cells leads phosphorylation on SMAD2 protein on serine 465/467 residue

Total-SMAD2 cellular assay validation on human and mouse cell lines

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, cell culture medium was removed and 50µL of lysis buffer were 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 Total-SMAD2 HTRF detection reagents were added. Signals were recorded overnight.

The Total-SMAD2 HTRF assay was able to detect human and mouse SMAD2 proteins.

total-SMAD2 cellular assay validation on human and mouse cell lines

HTRF assay compared to Western Blot using Total-SMAD2 cellular assay on mouse C2C12 cells

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 Total-SMAD2 cellular kit reagents. A side by side comparison showed the HTRF Total assay is at least 9-fold more sensitive than the Western Blot.

HTRF assay compared to Western Blot using total SMAD2 cellular assay on mouse C2C12 cells

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

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Species compatibility

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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.

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