The phospho-SMAD1 (Ser463/465) kit enables the cell-based quantitative detection of SMAD1 phosphorylated at Ser463/465, as a readout of the BMP/SMAD pathway.
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  • Ready-to-use Ready-to-use
  • High sensitivity High sensitivity
  • Faster and more convenient than ELISA Faster and more convenient than ELISA
  • Low sample consumption Low sample consumption
The phospho-SMAD1 (Ser463/465) kit enables the cell-based quantitative detection of SMAD1 phosphorylated at Ser463/465, as a readout of the BMP/SMAD pathway.
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Overview

This HTRF cell-based assay enables rapid, quantitative detection of SMAD1 phosphorylated at Serine 463/465. After phosphorylation and translocation to the nucleus, SMAD1 acts as a transcription factor in regulating the expression of genes involved in stem cell renewal, cell proliferation, apoptosis, migration, differentiation, and immune responses.

Benefits

  • SPECIFICITY
  • PRECISION

Phospho-SMAD1 (Ser463/465) assay principle

The Phospho-SMAD1 (Ser463/465) assay measures SMAD1 when phosphorylated at Ser463/465. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Phospho-SMAD1 (Ser463/465) assay uses 2 labeled antibodies: one with a donor fluorophore, the other one with an acceptor. The first antibody is selected for its specific binding to the phosphorylated motif on the protein, the second for its ability to recognize the protein independent 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.
Phospho-SMAD1 (Ser463/465) assay principle

Phospho-SMAD1 (Ser463/465) 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 phospho-SMAD1 (Ser463/465) HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Phospho-SMAD1 (Ser463/465) 2-plate assay protocol

Phospho-SMAD1 (Ser463/465) 1-plate assay protocol

Detection of Phosphorylated SMAD1 (Ser463/465) 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.
Phospho-SMAD1 (Ser463/465) 1-plate assay protocol

HTRF assay compared to Western Blot using phospho-Smad1 cellular assay on mouse C2C12 cells

Mouse C2C12 cells were cultured to 80% confluency. After BMP-4 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 kit reagents. A side by side comparison showed the HTRF Phospho assay is at least 8-fold more sensitive than the Western Blot, and shows optimal correlation.

HTRF assay compared to Western Blot using total SLP-76 cellular assays on human Jurkat cells
HTRF assay compared to Western Blot using total SLP-76 cellular assays on human Jurkat cells

Validation of the HTRF phospho-SMAD1 (Ser463/465) cellular assay on mouse and human cell lines

Cells were plated in a 96-well plate and incubated for 24h at 37°C, 5% COo2. After treatment for 30 min with BMP-4, cells were lysed, with 50 µL of lysis buffer added for 30min at RT under gentle shaking. 16 µL of lysate were transferred into a 384-well low volume white microplate and 4 µL of the HTRF phospho-SMAD1 (Ser463/465) detection reagents were added. The HTRF signal was recorded after a 4 hour incubation. Human cervical cancer HeLa cells were plated at 50,000 cells/ well and treated with human BMP-4. Mouse myoblast C2C12 cells were plated at 25,000 cells/ well and treated with mouse BMP-4.
Validation of the HTRF phospho-SMAD1 (Ser463/465) cellular assay on human HeLa cells
Validation of the HTRF phospho-SMAD1 (Ser463/465) cellular assay on mouse C2C12 cells

BMP/SMAD signaling pathway

BMP/SMAD signaling pathway plays a key role in embryogenesis, development, adult tissue homeostasis and immune responses. It is mediated by heterotetrameric complexes of BMPRI and BMPRI, that activate the intracellular signaling proteins SMAD1/5/8 by phosphorylating two C-terminal serine residues.

BMP-2/4 binding on BMPRI triggers the recruitment of BMPRII, which then transphosphorylates the intracellular domain of BMPRI.

BMPRI in turn phosphorylates SMAD1 at Ser463 and Ser465, enabling its oligomerization with the co-mediator (Co-SMAD) SMAD4. This complex translocates to the nucleus, where it functions as a transcription factor with coactivators and corepressors to regulate gene expression.

Inhibitory SMAD6 and SMAD7 are involved in feedback inhibition of the signaling pathway.

Signaling pathway of BMP/SMAD
Simplified pathway dissection with HTRF phospho-assays and CyBi-felix liquid handling

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

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

Cisbio starter packs

96-tests kits - Flyers

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

Do all cell-based kinase assays perform similarly? - 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

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

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

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

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

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