HTRF Human/Mouse Phospho-SMAD4 Thr277 Detection Kit HTRF®

The Phospho-SMAD4 (Thr277) kit enables the cell-based quantitative detection of SMAD4 phosphorylated on Thr277, as a readout of the TGFb pathway.
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  • All inclusive kit All inclusive kit
  • High sensitivity High sensitivity
  • Rapid Rapid
  • Low sample consumption Low sample consumption
The Phospho-SMAD4 (Thr277) kit enables the cell-based quantitative detection of SMAD4 phosphorylated on Thr277, as a readout of the TGFb pathway.


This HTRF cell-based assay enables the rapid, quantitative detection of SMAD4 phosphorylated at Threonine 277, as a readout of TGF-ß signaling activity.

TGF-ß receptors directly activate the SMAD2/3 complex by phosphorylation, inducing the association with phospho SMAD4 that then translocates to the nucleus and regulates the gene expression involved in apoptosis, migration, and differentiation, as well as in immune/inflammatory responses and extracellular matrix remodeling.



HTRF phospho SMAD4 Thr277 assay principle

The Phospho-SMAD4 (Thr277) assay measures SMAD4 when phosphorylated at Thr277. Unlike Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis, or transfer.

The Phospho-SMAD4 (Thr277) 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 involvingthe 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 Phospho T277 SMAD4 assay

Phospho-SMAD4 (Thr277) 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 additon of the Phospho-SMAD4 (Thr277) HTRF detection reagents.

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

Two-plate protocol of the HTRF Phospho T277 SMAD4 assay principle

Phospho-SMAD4 (Thr277) one-plate assay protocol

Detection of Phosphorylated SMAD4 (Thr277) 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.

One-plate protocol of the HTRF Phospho T277 SMAD4 assay principle

Phospho SMAD4 (Thr277) cell line compatibility

Hela and HAPI cell lines were selected to test human compatibility, while NIH 3T3 cells were chosen for mouse compatibility. 25,000, 50,000, and 100,000 cells of these different cell lines were plated in 96-well  culture plates. After a 48h incubation at 37°C, 5% CO2, the cell culture medium was removed and 50µL of lysis buffer were added to the wells. A lysis step was carried out, shaking gently for 30 minutes. 16µL of the pure samples diluted ½ were transferred into a 384-well small volume plate, then 4µL of Phospho T277 SMAD4 HTRF detection reagents were added. Signals were recorded overnight.

This data enables  better cell density selection for the different cell lines. The HTRF Phospho SMAD4 (Thr277) assay is able to detect human as well as mouse phosphorylated SMAD4.

cell line expression and density on Phospho T277 SMAD4 assay

Phospho SMAD4 (Thr277) assay specificity

HAPI wt were plated at 25,000 cells per well in a 96-well plate.

After an overnight incubation at 37°C, 5% CO2, the HAPI wt cells were treated with 25 nM of ON-TARGETplus siRNA (Horizon DIscovery) targeting specifically SMAD2, SMAD3, and SMAD4, or with a non-targeting siRNA (included as a control). After an overnight incubation at 37°C, 5% CO2, the medium was changed for a complete culture medium, and then the cells were incubated for an additional 24h at 37°C.

HAPI SMAD4 KO cells (Horizon Discovery)were plated at 25,000 cells per well in a 96-well plate and were incubated for 4 days.

After the incubation, the cells were lysed with 50 µL of supplemented lysis buffer #4 (1X), and 16 µL of lysates were transferred into a low volume white microplate before the addition of 4 µL of premixed HTRF Phospho T277 SMAD4 detection antibodies. The HTRF signal was recorded after an overnight incubation at RT.

Cell treatment with SMAD4 siRNA led to a significant downregulation of total SMAD4, leading to a 61% Phosphorylation decrease compared to the cells transfected with the non-targeting siRNA. Moreover, no HTRF signal was observed in the HAPI SMAD4 KO cell lysate . 

On the other hand, no decrease in signal was observed with cells treated with SMAD2 or SMAD3 SiRNA , demonstrating the specificity of the kit. The downregulation of SMAD2 & SMAD3 was checked using our HTRF Total SMAD2 & SMAD3 detection kits, showing a 60% and 66% loss of signal respectively.

HTRF Phospho SMAD4 (Thr277) Assay compared to Western Blot

SiRNA SMAD2 & 3 & 4 experiments on Phospho T277 SMAD4
SiRNA SMAD2 & 3 & 4 experiments on Phospho T277 SMAD4
SiRNA SMAD2 & 3 & 4 experiments on Phospho T277 SMAD4

HTRF Phospho SMAD4 (Thr277) Assay compared to Western Blot

HAPI cells were cultured in complete medium at 37°C, 5% CO2 in a T175 flask to 80% confluency.

The cells were lysed with 3 mL of supplemented lysis buffer #4 (1x) for 30 min at RT under gentle shaking. 

Serial dilutions of the cell lysate were performed using supplemented lysis buffer #4 (1x), and 16µL of pure sample and each dilution were transferred into a 384-well small volume microplate, before the addition of 4µL of Phospho T277-SMAD4 HTRF detection reagents. Signals were recorded overnight.

Equal amounts of lysates were loaded into a gel for a side by side comparison between HTRF and Western Blot.

In these conditions, the HTRF phospho SMAD4 (Thr277) assay is at least 4-fold more sensitive than the Western Blot.

Comparison of HTRF Phospho T277 SMAD4 kit with Western Blot

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

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

Lysis buffer compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - Flyers

Biomarker and Cell Signaling Assays for Fibrosis and NASH

HTRF and Alpha solutions for NASH - Flyers

Species compatibility

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

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

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

Best practices for analyzing tumor xenografts with HTRF phospho assays

Protocol for tumor xenograft analysis with HTRF - Technical Notes

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

HTRF Product Catalog

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

HTRF® cell signaling platform combined with iCell® Hepatocytes

A solution for phospho-protein analysis in metabolic disorders - 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

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

Product Insert SMAD4 T277 Kit / 64SMAD4T7PEG-64SMAD4T7PEH

64SMAD4T7PEG-64SMAD4T7PEH - Product Insert

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

Plate Reader Requirement

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