Smad2 (Phospho-Ser467)Colorimetric Cell-Based ELISA Kit (CBCAB00136)

Product Name:	Smad2 (Phospho-Ser467)Colorimetric Cell-Based ELISA Kit
Product Code:	CBCAB00136
ELISA Type:	Cell-Based
Target:	Smad2 (Phospho-Ser467)
Reactivity:	Human, Mouse, Rat
Dynamic Range:	> 5000 Cells
Detection Method:	Colorimetric 450 nm
Format:	2 x 96-Well Microplates

The Smad2 (Phospho-Ser467) Colorimetric Cell-Based ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can detect Smad2 protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated Smad2 in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on Smad2 phosphorylation.

Qualitative determination of Smad2 (Phospho-Ser467) concentration is achieved by an indirect ELISA format. In essence, Smad2 (Phospho-Ser467) is captured by Smad2 (Phospho-Ser467)-specific primary antibodies while the HRP-conjugated secondary antibodies bind the Fc region of the primary antibody. Through this binding, the HRP enzyme conjugated to the secondary antibody can catalyze a colorimetric reaction upon substrate addition. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1.	A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target absorbance values.
2.	Following the colorimetric measurement of HRP activity via substrate addition, the Crystal Violet whole-cell staining method may be used to determine cell density. After staining, the results can be analysed by normalizing the absorbance values to cell amounts, by which the plating difference can be adjusted.

Database Information:	Gene ID: 4087, UniProt ID: Q15796, OMIM: 601366, Unigene: Hs.12253/Hs.705764
Gene Symbol:	SMAD2
Sub Type:	Phospho

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

Component	Quantity
96-Well Cell Culture Clear-Bottom Microplate	2 plates
10X TBS	24 mL
Quenching Buffer	24 mL
Blocking Buffer	50 mL
15X Wash Buffer	50 mL
Primary Antibody Diluent	12 mL
100x Anti-Phospho Target Antibody	60 µL
100x Anti-Target Antibody	60 µL
Anti-GAPDH Antibody	60 µL
HRP-Conjugated Anti-Rabbit IgG Antibody	12 mL
HRP-Conjugated Anti-Mouse IgG Antibody	12 mL
SDS Solution	12 mL
Stop Solution	24 mL
Ready-to-Use Substrate	12 mL
Crystal Violet Solution	12 mL
Adhesive Plate Seals	2 seals

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

Microplate reader able to measure absorbance at 450 nm and/or 595 nm for Crystal Violet Cell Staining (Optional)
Micropipettes with capability of measuring volumes ranging from 1 µL to 1 ml
37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
Graph paper or computer software capable of generating or displaying logarithmic functions
Absorbent papers or vacuum aspirator
Test tubes or microfuge tubes capable of storing ≥1 ml
Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
Orbital shaker (optional)
Deionized or sterile water

*Note: Protocols are specific to each batch/lot. For the correct instructions please follow the protocol included in your kit.

Step	Procedure
1.	Seed 200 µL of 20,000 adherent cells in culture medium in each well of a 96-well plate. The plates included in the kit are sterile and treated for cell culture. For suspension cells and loosely attached cells, coat the plates with 100 µL of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.	Incubate the cells for overnight at 37°C, 5% CO2.
3.	Treat the cells as desired.
4.	Remove the cell culture medium and rinse with 200 µL of 1x TBS, twice.
5.	Fix the cells by incubating with 100 µL of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.	Remove the Fixing Solution and wash the plate 3 times with 200 µL 1x Wash Buffer for five minutes each time with gentle shaking on the orbital shaker. The plate can be stored at 4°C for a week.
7.	Add 100 µL of Quenching Buffer and incubate for 20 minutes at room temperature.
8.	Wash the plate 3 times with 1x Wash Buffer for 5 minutes each time.
9.	Add 200 µL of Blocking Buffer and incubate for 1 hour at room temperature.
10.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
11.	Add 50 µL of 1x primary antibodies Anti-Smad2 (Phospho-Ser467) Antibody, Anti-Smad2 Antibody and/or Anti-GAPDH Antibody) to the corresponding wells, cover with Parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
13.	Add 50 µL of 1x secondary antibodies (HRP-Conjugated AntiRabbit IgG Antibody or HRP-Conjugated Anti-Mouse IgG Antibody) to corresponding wells and incubate for 1.5 hours at room temperature.
14.	Wash 3 times with 200 µL of 1x Wash Buffer for 5 minutes each time.
15.	Add 50 µL of Ready-to-Use Substrate to each well and incubate for 30 minutes at room temperature in the dark.
16.	Add 50 µL of Stop Solution to each well and read OD at 450 nm immediately using the microplate reader.

(Additional Crystal Violet staining may be performed if desired – details of this may be found in the kit technical manual.)