SMAD1 (pS463/465) PhosphoTracer ELISA Kit (ab119647)

Overview

  • Product nameSMAD1 (pS463/465) PhosphoTracer ELISA KitSee all Smad 1 (phospho Ser463/465) kits ...
  • Detection methodFluorescent
  • Tests
    1 x 96 well plate
  • Sample type
    Cell culture extracts
  • Assay typeSemi-quantitative
  • Sensitivity
    5000 cells/well
  • Range
    5000 cells/well - 100000 cells/well
  • Assay time
    2h 0m
  • Assay durationOne step assay
  • Species reactivity
    Reacts with: Mouse, Human
    Predicted to work with: Rat
  • Product overview

    PhosphoTracer assays use a traditional immuno-sandwich format, but with a major difference both the analyte and the assay reagents are added to the PhosphoTracer assay microplate at the same time. After a short incubation period, unbound assay reagents and analytes are washed away, and immuno-complexes containing both antibodies are detected. The process can take as little as 60 minutes to complete.

    PhosphoTracer kits also allow a higher degree of assay flexibility. In contrast to other ELISA formats, no antibodies are present on the assay microplate itself, so assays for several different targets can be performed in different wells on the same microplate. Simply mix the lysate with your target reagents of choice, using the microplate configuration of your choice.

    A whole new way of performing cellular assays, PhosphoTracer takes the hard work out of running a standard ELISA, while still giving the high quality results expected from a sandwich immunoassay. Fully self-contained kits are supplied in convenient 96-well packs. Simple to use and highly sensitive PhosphoTracer kits are designed to get results, fast.

     

    Abcam's PhosphoTracer SMAD1 (pSer463/465) assay kits detect endogenous levels of SMAD1 (GenBank Accession NP_001003688) in cellular lysates. Phospho-SMAD1 assay kits only detect SMAD1 when phosphorylated at Ser463/465. Total SMAD1 assay kits detect SMAD1 irrespective of phosphorylation status. Based on sequence similarity, cross reaction to SMAD5 and SMAD8 may occur.



    The substrate used with the HRP conjugated detection antibody is a combination of 10-Acetyl-3,7-dihydroxyphenoxazine (ADHP) (wavelength exc/em = 530-540nm / 590-600nm), a highly sensitive and stable substrate for HRP) and ADHP Dilution Buffer (a stabilized H2O2 solution).
    Learn more about the fluorogenic substrate, ADHP.

  • Tested applicationsSandwich ELISA more details
  • PlatformMicroplate

Properties

  • FunctionTranscriptional modulator activated by BMP (bone morphogenetic proteins) type 1 receptor kinase. SMAD1 is a receptor-regulated SMAD (R-SMAD). SMAD1/OAZ1/PSMB4 complex mediates the degradation of the CREBBP/EP300 repressor SNIP1.
  • Tissue specificityUbiquitous. Highest expression seen in the heart and skeletal muscle.
  • Sequence similaritiesBelongs to the dwarfin/SMAD family.
    Contains 1 MH1 (MAD homology 1) domain.
    Contains 1 MH2 (MAD homology 2) domain.
  • Post-translational
    modifications
    Phosphorylated on serine by BMP type 1 receptor kinase.
    Ubiquitin-mediated proteolysis by SMAD-specific E3 ubiquitin ligase SMURF1.
  • Cellular localizationCytoplasm. Nucleus. Cytoplasmic in the absence of ligand. Migrates to the nucleus when complexed with SMAD4. Co-localizes with LEMD3 at the nucleus inner membrane.
  • Target information above from: UniProt accession Q15797 The UniProt Consortium
    The Universal Protein Resource (UniProt) in 2010
    Nucleic Acids Res. 38:D142-D148 (2010) .

    Information by UniProt
  • Alternative names
    • BSP-1
    • BSP1
    • HsMAD1
    • JV4-1
    • JV41
    • MAD homolog 1
    • MAD mothers against decapentaplegic homolog 1
    • Mad-related protein 1
    • MADH1
    • MADR1
    • Mothers against decapentaplegic homolog 1
    • Mothers against DPP homolog 1
    • SMAD 1
    • SMAD family member 1
    • SMAD mothers against DPP homolog 1
    • Smad1
    • SMAD1_HUMAN
    • TGF beta signaling protein 1
    • Transforming growth factor-beta-signaling protein 1
    see all
  • Database links

Applications

Our Abpromise guarantee covers the use of ab119647 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Notes
Sandwich ELISA Use at an assay dependent concentration. Tested in HeLa, C2C12.

SMAD1 (pS463/465) PhosphoTracer ELISA Kit images

  • Using the SMAD1 assay kits or Western blot, SMAD1 phosphorylation at Ser463/465 is detected in BMP4-treated C2C12 cells (+), compared with C2C12 cells treated with 50 µM dorsomorphin (-). Upon phosphorylation, SMAD1 levels decrease as SMAD1 becomes ubiquitinated.
  • C2C12 cells were seeded at 5K cells/well in a 96 well tissue culture microplate overnight. The next day cells were serum starved for 60 minutes, then treated with various concentrations of dorsomorphin for 30 mins. Subsequently, the cells stimulated with 50 ng/mL BMP-4 for 30 min. The medium was removed from the wells, and cells were lysed with 120 µl/well of Lysis Mix, with shaking for 10 min. The lysates were transferred to a PhosphoTracer assay plate and assayed for either total or phospho-SMAD1, using the standard protocol. Signal in the wells was determined using a plate reader.

Protocols

References for SMAD1 (pS463/465) PhosphoTracer ELISA Kit (ab119647)

ab119647 has not yet been referenced specifically in any publications.

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The stop solution is not an acid because acid is incompatible with the fluorescent product generated here. Furthermore, it is important to emphasize that only 10 microlitres of stop solution per ...

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1. There is no particular reason why protease inhibitors are not included in the lysis buffer. Your customer could try any of the common protease inhibitors that they would normally add to their extraction buffer to see if their results are improved. P...

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