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Total SMAD1 cellular kit HTRF®

The Total SMAD1 kit detects cellular SMAD1 and can be used as a normalization assay with our phospho-SMAD1 kit to assist in optimal monitoring of BMP/SMAD signaling.
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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 Total SMAD1 kit detects cellular SMAD1 and can be used as a normalization assay with our phospho-SMAD1 kit to assist in optimal monitoring of BMP/SMAD signaling.
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Overview

The total SMAD1 cellular assay kit is designed to monitor the expression level of SMAD1, whether phosphorylated or unphosphorylated. It is compatible with our Phospho-SMAD1 kit, and enables the analysis of phosphorylated and total proteins from a single sample for better studies of BMP/SMAD signaling.

Benefits

  • SPECIFICITY
  • PRECISION
  • DATA NORMALIZATION

Total-SMAD1 assay principle

The Total-SMAD1 assay quantifies the expression level of SMAD1 in a cell lysate. Contrary to Western Blot, the assay is entirely plate-based and does not require gels, electrophoresis or transfer. The Total-SMAD1 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 SMAD1 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-SMAD1 assay principle

Total-SMAD1 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-SMAD1 HTRF detection reagents. This protocol enables the cells' viability and confluence to be monitored.
Total-SMAD1 2-plate assay protocol

Total-SMAD1 1-plate assay protocol

Detection of total SMAD1 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-SMAD1 1-plate assay protocol

Validation of the HTRF total SMAD1 cellular assay on mouse and human cell lines

Cells were cultured until 80% confluency in a T175 flask with complete culture medium at 37°C, 5% CO2 (2 days). Cells are treated for 30 min with 200 ng/mL BMP-4, then lysed 30min at RT and under gentle shaking with 3 mL of supplemented lysis buffer #4. Soluble supernatants were collected after a 10 minute centrifugation. Serial dilutions of the cell lysate were performed in the supplemented lysis buffer and 16 µL of each dilution were transferred into a 384-well low volume white microplate before adding 4 µL of the HTRF total SMAD1 detection reagents. HTRF signal was recorded after incubating overnight. Human cervical cancer HeLa cells and mouse myoblast C2C12 cells were treated with human BMP-4 and mouse BMP-4 respectively.
Validation of total SMAD1 on human HeLa cells
Validation of total SMAD1 on mouse 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.

BMP/SMAD 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

Lysis buffer compatibility

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

HTRF cellular phospho-protein assays

Physiologically relevant results fo fast flowing research - 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

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

Product Insert SMAD1 total Kit / 63ADK063PEG-63ADK063PEH

63ADK063PEG-63ADK063PEH - Product Insert

HTRF Product Catalog

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

Insight into fibrosis development and signaling pathways

A comprehensive overview of fibrosis development - Guides

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

Methodological Aspects of Homogeneous Time-Resolved Fluorescence (HTRF)

Learn how to reduce time and sample consumption - Application Notes

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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