Human Phospho-SMAD3 (S423/425) ELISA

The RayBio® Human Phospho-SMAD3 (S423/425) ELISA Kit semi-quantitatively measures phosphorylated SMAD3 (Ser423/425) in lysate samples.

$521.00  
PEL-SMAD3-S423-1
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Assay Format

Pathway:
  • Angiogenesis
  • Cell Cycle
  • Stem Cell
  • TGF-beta
Specificity:
The antibody pair provided in this kit recognizes human SMAD3 phosphorylated at site Serine-423/425.
Number of Targets Detected:
1
Species Detected:
  • Human
Compatible Sample Types:
  • Cell Lysates
  • Tissue Lysates
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
  • Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Protein Information

Accession Number:
  • P84022
Gene Symbols:
  • MADH3
  • SMAD3
Protein Name & Synonyms:
Mothers against decapentaplegic homolog 3 (MAD homolog 3) (Mad3) (Mothers against DPP homolog 3) (hMAD-3) (JV15-2) (SMAD family member 3) (SMAD 3) (Smad3) (hSMAD3)

Introduction

RayBio® Phospho-SMAD3 (Ser423/425) ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates. By determining phosphorylated SMAD3 protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.

This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human phospho-SMAD3. An anti-pan SMAD3 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and SMAD3 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-SMAD3 (Ser423/425) antibody is used to detect phosphorylated SMAD3. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of SMAD3 (Ser423/425) bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

  • Rapidly measure phosphorylated protein in lysates
  • Screen numerous different cell lysates without performing a Western Blot analysis
  • Minimal hands-on time, convenient, and non-radioactive material

Application Notes

Kit Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Anti-Phospho Antibody
  • HRP-Conjugated Secondary Antibody
  • Assay Diluent
  • TMB One-Step Substrate
  • Stop Solution
  • Lysis Buffer
  • Positive Control Sample
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Protease and Phosphatase inhibitors
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Benchtop rocker or shaker
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Positive Control
A549 cells were treated with Calyculin A and PMA. Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
SMAD3 (Ser423/425) - Positive Control
Calyculin A and PMA Stimulation of A549 Cell Line
A549 cells were treated with Calyculin A and PMA. Cell lysates were analyzed using this phosphoELISA and Western Blot.
SMAD3 (Ser423/425) - ELISA Kit
SMAD3 (Ser423/425) - Western Blot

Storage/Stability

Upon receipt, the kit should be stored at -20°C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4°C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at -20°C. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.