SMAD1 (pS463/S465 + Total SMAD1) ELISA Kit (RMEK-0155973)
Cat. No.: RMEK-0155973
Category: ELISA Kits
INQUIRY
1 x 96 tests
SMAD1 (pS463/S465) and SMAD1 (Total) in vitro ELISA kit is designed for the semi-quantitative measurement of SMAD (pS463/S465) and Total SMAD1 protein in human and mouse cells.
The ELISA employs a labeled capture and detector antibody which immunocaptures the sample analyte in solution. This entire complex (capture antibody/protein/detector antibody) is in turn immobilized in the well by immunoaffinity via the anti-tag antibody. Samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material; the TMB substrate is then added. The reaction is stopped by addition of Stop Solution which stops the color development and completes any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
ASSAY SPECIFICITY The SMAD1 (pS463/S465) assay detects endogenous levels of SMAD1 in cellular lysates, only when phosphorylated at Ser463/465. Based on sequence similarity, cross reaction to SMAD5 and SMAD8 may occur.
The SMAD1 (Total) assay detects endogenous levels of SMAD1 in cellular lysates. Total SMAD1 assay kits detect SMAD1, irrespective of phosphorylation status. Based on sequence similarity, cross reaction to SMAD5 and SMAD8 may occur.
SPECIES REACTIVITY This kit detects SMAD1 (pS463/S465) and SMAD1 (Total) in Human and mouse cell culture extracts. Detection in rat samples is also expected. Other species should be tested on a case-by-case basis.
The ELISA employs a labeled capture and detector antibody which immunocaptures the sample analyte in solution. This entire complex (capture antibody/protein/detector antibody) is in turn immobilized in the well by immunoaffinity via the anti-tag antibody. Samples or standards are added to the wells, followed by the antibody mix. After incubation, the wells are washed to remove unbound material; the TMB substrate is then added. The reaction is stopped by addition of Stop Solution which stops the color development and completes any color change from blue to yellow. Signal is generated proportionally to the amount of bound analyte and the intensity is measured at 450 nm. Optionally, instead of the endpoint reading, development of TMB can be recorded kinetically at 600 nm.
ASSAY SPECIFICITY The SMAD1 (pS463/S465) assay detects endogenous levels of SMAD1 in cellular lysates, only when phosphorylated at Ser463/465. Based on sequence similarity, cross reaction to SMAD5 and SMAD8 may occur.
The SMAD1 (Total) assay detects endogenous levels of SMAD1 in cellular lysates. Total SMAD1 assay kits detect SMAD1, irrespective of phosphorylation status. Based on sequence similarity, cross reaction to SMAD5 and SMAD8 may occur.
SPECIES REACTIVITY This kit detects SMAD1 (pS463/S465) and SMAD1 (Total) in Human and mouse cell culture extracts. Detection in rat samples is also expected. Other species should be tested on a case-by-case basis.
Product Features
| Species Reactivity | Mouse, Human |
|---|---|
| Detection Method | Colorimetric |
| Assay Duration | One step assay |
| Assay Time | 1.5 h |
| Assay Type | Semi-quantitative |
| Precision | Intra-Assay-PS463/S465-6-2.3%; Intra-Assay-Total-6-5.3%; Inter-Assay-PS463/S465-3-7.5%; Inter-Assay-Total-3-5.6% |
| Sensitivity | 20 µg/mL |
| Range | 10 µg/mL - 1000 µg/mL |
| Sample Type | Cell Lysate, Tissue Homogenate |
| Key Features | One-wash 90 minute protocol; Sensitivity: 20 µg/mL; Range: 10 µg/mL - 1000 µg/mL; Sample type: Cell Lysate, Tissue Homogenate; Detection method: Colorimetric; Assay type: Semi-quantitative; Reacts with: Mouse, Human |
Target Information
| Target Symbol | SMAD1 |
|---|---|
| UniProt ID | Q15797 |
| Function | Transcriptional modulator that plays a role in various cellular processes, including embryonic development, cell differentiation, and tissue homeostasis. Upon BMP ligand binding to their receptors at the cell surface, is phosphorylated by activated type I BMP receptors (BMPRIs) and associates with SMAD4 to form an heteromeric complex which translocates into the nucleus acting as transcription factor. In turn, the hetero-trimeric complex recognizes cis-regulatory elements containing Smad Binding Elements (SBEs) to modulate the outcome of the signaling network. SMAD1/OAZ1/PSMB4 complex mediates the degradation of the CREBBP/EP300 repressor SNIP1. Positively regulates BMP4-induced expression of odontogenic development regulator MSX1 following IPO7-mediated nuclear import (By similarity). |
| Cellular Localization | Cytoplasm. Nucleus. Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4. Co-localizes with LEMD3 at the nucleus inner membrane. Exported from the nucleus to the cytoplasm when dephosphorylated (By similarity). |
| Domain | The MH2 domain mediates phosphorylation-dependent trimerization through L3 loop binding of phosphoserines in the adjacent subunit. |
| Post-transcriptional Modifications | Phosphorylation of the C-terminal SVS motif by BMP type 1 receptor kinase activates SMAD1 by promoting dissociation from the receptor and trimerization with SMAD4. Phosphorylation by ERK2 MAP kinase in response to EGF or HGF prevents SMAD1 nuclear accumulation and transcriptional activity in response to BMP. Dephosphorylation, probably by PPM1A, induces its export from the nucleus to the cytoplasm (By similarity). Dephosphorylation is inhibited by association with EGR1 (By similarity). Phosphorylation by CDK8/9 creates binding sites for YAP1, and subsequent phosphorylation by GSK3 switches off YAP1 binding and adds binding sites for SMURF1. Ubiquitinated by SMAD-specific E3 ubiquitin ligase SMURF1, leading to its degradation. Monoubiquitinated, leading to prevent DNA-binding. Deubiquitination by USP15 alleviates inhibition and promotes activation of TGF-beta target genes. Dephosphorylation, probably by PPM1A, induces its export from the nucleus to the cytoplasm (By similarity). Phospho-SMAD1 is ubiquitinated by CHIP leading to disruption of the SMAD1-SMAD4 complex. |
| Involvement in Disease | SMAD1 variants may be associated with susceptibility to pulmonary hypertension, a disorder characterized by plexiform lesions of proliferating endothelial cells in pulmonary arterioles. The lesions lead to elevated pulmonary arterial pression, right ventricular failure, and death. The disease can occur from infancy throughout life and it has a mean age at onset of 36 years. Penetrance is reduced. Although familial pulmonary hypertension is rare, cases secondary to known etiologies are more common and include those associated with the appetite-suppressant drugs. |
Storage & Shipping
| Storage | Store at 2-8°C |
|---|---|
| Shipping | Gel Packs |
| Stability | The stability of kit is determined by the loss rate of activity. The loss rate of this kit is less than 5% within the expiration date under appropriate storage condition. |
For research use only. Not for clinical use.
