Human Smad2/MADH2(Mothers Against Decapentaplegic Homolog 2)ELISA Kit (HUES03594)



ELISA Kit Technical ManualMSDS

Human Smad2/MADH2(Mothers Against Decapentaplegic Homolog 2)ELISA Kit

This ELISA kit uses Sandwich-ELISA as the method. The micro ELISA plate provided in this kit has been pre-coated with an antibody specific to Human Smad2/MADH2 . Standards or samples are added to the appropriate micro ELISA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human Smad2/MADH2 and Avidin-Horseradish Peroxidase (HRP) conjugate are added to each micro plate well successively and incubated. Free components are washed away. The substrate solution is added to each well. Only those wells that contain Human Smad2/MADH2, biotinylated detection antibody and Avidin-HRP conjugate will appear blue in color. The enzyme-substrate reaction is terminated by adding Stop Solution and the color turns yellow. The optical density (OD) is measured spectrophotometrically at a wavelength of 450 nm ± 2 nm. The OD value is proportional to the concentration of Human Smad2/MADH2. The concentration of Human Smad2/MADH2 in samples can be calculated by comparing the OD of the samples to the standard curve.

Assay typeSandwich
Assay time4.5h
Detection MethodColormetric
Detection Range78.13-5000 pg/mL
Sensitivity46.88 pg/mL
Sample Volume Required Per Well100uL
Sample TypeSerum, plasma and other biological fluids


This kit recognizes Human Smad2/MADH2 in samples. No significant cross-reactivity or interference between Human Smad2/MADH2 and analogues was observed.

Typical Data

As the OD values of the standard curve may vary according to the conditions of the actual assay performance (e.g. operator, pipetting technique, washing technique or temperature effects), the operator should establish a standard curve for each test. Typical standard curve and data is provided below for reference only.

O.D Average Corrected


Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Smad2/MADH2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Smad2/MADH2 were tested on 3 different plates, 20 replicates in each plate.

Intra-assay Precision Inter-assay Precision
Mean (pg/mL)255.50767.172141.19269.86816.071957.09
Standard deviation13.0838.5187.5718.0533.4682.78
C V (%)


The recovery of Human Smad2/MADH2 spiked at three different levels in samples throughout the range of the assay was evaluated in various matrices.

Sample Type Range (%) Average Recovery (%)
Serum (n=5)88-10095
EDTA plasma (n=5)91-10497
Cell culture media (n=5)97-111102


Samples were spiked with high concentrations of Human Smad2/MADH2 and diluted with Reference Standard & Sample Diluent to produce samples with values within the range of the assay.

Serum (n=5) EDTA plasma (n=5) Cell culture media (n=5)
1:2Range (%)91-10794-10995-107
Average (%)98101100
1:4Range (%)86-10080-9283-97
Average (%)938589
1:8Range (%)91-10582-9686-101
Average (%)968793
1:16Range (%)89-10481-9583-95
Average (%)968888

Kit Components & Storage

An unopened kit can be stored at 4'C for 1 month. If the kit is not used within 1 month, store the items separately according to the following conditions once the kit is received.

Item Specifications Storage
Micro ELISA Plate(Dismountable)8 wells X12 strips-20'C, 6 months
Reference Standard2 vials
Concentrated Biotinylated Detection Ab (100X)1 vial, 120 uL
Concentrated HRP Conjugate (100X)1 vial, 120 uL-20'C(shading light), 6 months
Reference Standard & Sample Diluent1 vial, 20 mL4'C, 6 months
Biotinylated Detection Ab Diluent1 vial, 14 mL
HRP Conjugate Diluent1 vial, 14 mL
Concentrated Wash Buffer (25X)1 vial, 30 mL
Substrate Reagent1 vial, 10 mL4'C(shading light)
Stop Solution1 vial, 10 mL4'C
Plate Sealer5 pieces
Product Description1 copy
Certificate of Analysis1 copy

Human Smad2/MADH2(Mothers Against Decapentaplegic Homolog 2)ELISA Kit (HUES03594) Assay procedure

    1. Set standard, test sample and control (zero) wells on the pre-coated plate and record theirpositions. It is recommended to measure each standard and sample in duplicate. Note: addall solutions to the bottom of the plate wells while avoiding contact with the well walls. Ensuresolutions do not foam when adding to the wells.
    2. Aliquot 100µl of standard solutions into the standard wells.
    3. Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
    4. Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids) into test sample wells.
    5. Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
    6. Aspirate the liquid from each well, do not wash. Immediately add 100µL of BiotinylatedDetection Ab working solution to each well. Cover the plate with a plate seal and gently mix.Incubate for 1 hour at 37°C.
    7. Aspirate or decant the solution from the plate and add 350µL of wash buffer to each welland incubate for 1-2 minutes at room temperature. Aspirate the solution from each well andclap the plate on absorbent filter paper to dry. Repeat this process 3 times. Note: a microplatewasher can be used in this step and other wash steps.
    8. Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
    9. Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
    10. Add 90µL of Substrate Reagent to each well. Cover with a new plate seal and incubate forapproximately 15 min at 37°C. Protect the plate from light. Note: the reaction time can beshortened or extended according to the actual color change, but not by more than 30min.
    11. Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
    12. Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.

Human Smad2/MADH2(Mothers Against Decapentaplegic Homolog 2)ELISA Kit (HUES03594) Protein Information

UniProt Protein Function:SMAD2: ubiquitously expressed transcription factor phosphorylated and activated by TGF-beta receptor-type kinases. Participates in a wide range of critical processes including morphogenesis, cell-fate determination, proliferation, differentiation and apoptosis. Phosphorylated Smads dimerize with collaborating Smad4 and are translocated into the nucleus, where the transcription of target genes is stimulated. Two alternatively spliced isoforms have been described.
UniProt Protein Details:

Protein type:Transcription factor; DNA-binding

Chromosomal Location of Human Ortholog: 18q21.1

Cellular Component: nucleoplasm; transcription factor complex; nuclear chromatin; cytoplasm; nucleus; cytosol

Molecular Function:transcription activator binding; metal ion binding; transcription factor binding; transforming growth factor beta receptor, pathway-specific cytoplasmic mediator activity; phosphatase binding; protein binding; DNA binding; ubiquitin protein ligase binding; double-stranded DNA binding; chromatin binding; SMAD binding; transforming growth factor beta receptor binding; transcription factor activity

Biological Process: developmental growth; somatic stem cell maintenance; positive regulation of transcription, DNA-dependent; activin receptor signaling pathway; paraxial mesoderm morphogenesis; gastrulation; palate development; primary microRNA processing; negative regulation of transcription from RNA polymerase II promoter; post-embryonic development; anterior/posterior pattern formation; negative regulation of cell proliferation; regulation of transforming growth factor beta receptor signaling pathway; ureteric bud development; transforming growth factor beta receptor signaling pathway; embryonic foregut morphogenesis; pancreas development; response to glucose stimulus; positive regulation of BMP signaling pathway; transcription initiation from RNA polymerase II promoter; pericardium development; cell fate commitment; regulation of binding; transcription, DNA-dependent; in utero embryonic development; common-partner SMAD protein phosphorylation; embryonic cranial skeleton morphogenesis; SMAD protein complex assembly; organ growth; zygotic determination of dorsal/ventral axis; mesoderm formation; endoderm formation; insulin secretion; gene expression; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transforming growth factor beta receptor signaling pathway; negative regulation of transcription, DNA-dependent; lung development

NCBI Summary:The protein encoded by this gene belongs to the SMAD, a family of proteins similar to the gene products of the Drosophila gene 'mothers against decapentaplegic' (Mad) and the C. elegans gene Sma. SMAD proteins are signal transducers and transcriptional modulators that mediate multiple signaling pathways. This protein mediates the signal of the transforming growth factor (TGF)-beta, and thus regulates multiple cellular processes, such as cell proliferation, apoptosis, and differentiation. This protein is recruited to the TGF-beta receptors through its interaction with the SMAD anchor for receptor activation (SARA) protein. In response to TGF-beta signal, this protein is phosphorylated by the TGF-beta receptors. The phosphorylation induces the dissociation of this protein with SARA and the association with the family member SMAD4. The association with SMAD4 is important for the translocation of this protein into the nucleus, where it binds to target promoters and forms a transcription repressor complex with other cofactors. This protein can also be phosphorylated by activin type 1 receptor kinase, and mediates the signal from the activin. Alternatively spliced transcript variants have been observed for this gene. [provided by RefSeq, May 2012]
UniProt Code:Q15796
NCBI GenInfo Identifier:13633914
NCBI Gene ID:4087
NCBI Accession:Q15796.1
UniProt Related Accession:Q15796
Molecular Weight:52kDa
NCBI Full Name:Mothers against decapentaplegic homolog 2
NCBI Synonym Full Names:SMAD family member 2
NCBI Official Symbol:SMAD2  
NCBI Official Synonym Symbols:JV18; MADH2; MADR2; JV18-1; hMAD-2; hSMAD2  
NCBI Protein Information:mothers against decapentaplegic homolog 2; MAD homolog 2; mother against DPP homolog 2; Sma- and Mad-related protein 2; SMAD, mothers against DPP homolog 2
UniProt Protein Name:Mothers against decapentaplegic homolog 2
UniProt Synonym Protein Names:JV18-1; Mad-related protein 2; hMAD-2; SMAD family member 2; SMAD 2; Smad2; hSMAD2
Protein Family:Mothers against decapentaplegic
UniProt Gene Name:SMAD2  
UniProt Entry Name:SMAD2_HUMAN
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