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Phospho-SMAD1 (Ser463/465) & Total SMAD1 Cellular Assay Kits   Phospho- and total SMAD1 assays for studying SMAD1 in BMP/SMAD signaling Cisbio's cell-based homogeneous HTRF® phospho- and total SMAD1 assays enable quantitative detection of the modulation of SMAD1 phosphorylated on Serine 463/465. After BMP-2/4 binding on the complexes of BMP type I and type II kinase receptors, SMAD1 is phosphorylated on Ser463/465 by the BMP type I receptor, then translocates to the nucleus. There, it functions as a transcription factor in regulating the expression of genes involved in stem cell renewal, cell proliferation, apoptosis, migration, differentiation and immune responses. Phospho- and total SMAD1 assays are therefore valuable tools in oncology, cardiovascular and inflammation research. With a streamlined protocol without any washing steps and their adaptability to low-volume formats, the phospho- and total SMAD1 assays can be used from basic research up to preclinical drug discovery phases. The kits contain all the reagents you need, and offer increased throughput compared to ELISA or Western Blot.    Features Cell-based sandwich immunoassays Validated on endogenous levels of human and mouse cell lines (HeLa and C2C12) Low-volume samples required Truly homogeneous, simple protocol with no wash steps More convenient and faster than ELISA or WB   Applications Quantification of phospho-SMAD1 Ser463/465 Functional screening of BMP/SMAD signaling pathway modulators Total SMAD1 assay for normalization   Assay Principle  HTRF® - the homogeneous cell-based sandwich immunoassay   Cisbio Bioassay’s phospho- and total SMAD1 assays are based on a TR-FRET sandwich immunoassay format comprising two specific anti-SMAD1 antibodies, one labeled with Eu3+-cryptate (donor) and the other with d2 (acceptor). The antibodies specifically bind to SMAD1, and the proximity of the donor and the acceptor then leads to a fluorescent TR-FRET signal. The protocol is optimized for a 384-well plate format, but can easily be further miniaturized or upscaled. Only low sample volumes are needed. The detection reagents may be pre-mixed and added in a single dispensing step for direct detection. No washing is needed at any step.   The assays can be run with frozen cell lysates or fresh cells in culture. After cell lysis, endogenous phospho- or total-SMAD1 can be quantitatively detected using the HTRF phospho-SMAD1 (Ser463/465) and total SMAD1 cellular assay kit reagents and most TR-FRET multimode plate readers.   A simpler, more flexible assay protocol - adapted to your applications Two-plate assay protocol   For added flexibility, the assay can be run under a two-plate assay protocol, where cells are plated, treated and lysed in a 96-well culture plate. For detection, lysates are subsequently transferred to a 384 low volume assay plate where the HTRF® reagents are added. This also enables monitoring of the cells' viability and confluence in an appropriate cell culture plate.     Product performance 1. 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.             2. HTRF® assay compared to Western Blot  Mouse C2C12 cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2 until 80% confluency was reached. After treatment for 30 min with 200 ng/mL mouse BMP-4 the cells were lysed, with 3 mL of supplemented lysis buffer#4 added for 30min at RT under gentle shaking. 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 the addition of 4 µL of the HTRF phospho-SMAD1 (Ser463/465) detection reagents. Equal amounts of lysates were used for a side by side comparison of Western Blot and HTRF®.   Using HTRF® phospho-SMAD1 (Ser463/465), just 800 cells were sufficient for minimal signal detection, while 6,300 cells weare needed for a Western Blot signal detection. The HTRF® assay is at least 8-fold more sensitive than the Western Blot, and shows optimal correlation.         3. Validation of the HTRF total SMAD1 cellular assay on mouse and human cell lines Cells were seeded in a T175 flask in complete culture medium and incubated for 2 days at 37°C, 5% CO2 until 80% confluency was reached. After treatment for 30 min with 200 ng/mL BMP-4, cells were lysed, with 3 mL of supplemented lysis buffer #4 added for 30min at RT under gentle shaking. 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 the addition of 4 µL of the HTRF phosphtotal-SMAD1 (Ser463/465) detection reagents. The HTRF signal was recorded after an overnight incubation.   Human cervical cancer HeLa cells were treated with human BMP-4.       Mouse myoblast C2C12 cells were treated with mouse BMP-4.     Simplified pathway BMP/SMAD signaling pathway Bone Morphogenetic Proteins (BMPs) belong to the Transforming Growth Factor-β (TGF-β) superfamily which has ~ 20 different members.    BMP signaling is mediated by heterotetrameric complexes of BMP type I and type II serine/threonine kinase receptors (BMPRI, also called Alk and BMPRII), that directly activate the intracellular signaling proteins SMAD1/5/8 by phosphorylation at 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.   SMAD1 is also known as mothers against decapentaplegic homolog 1 (MADH1).   The BMP/SMAD signaling pathway plays a key role in embryogenesis, development, adult tissue homeostasis and immune responses.          

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