Human SMAD2 ELISA Kit (HUFI01809)

Product Name:	Human SMAD2 ELISA Kit
Product Code:	HUFI01809
Size:	96 Assays
Alias:	SMAD2, JV18, MADH2, MADR2, JV18-1, hMAD-2, hSMAD2, SMAD family member 2, SMAD 2, Smad2, hSMAD2, JV18-1, Mad-related protein 2, hMAD-2
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human SMAD2 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.188ng/ml
Range:	0.313-20ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human SMAD2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human SMAD2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	87-95	91
EDTA plasma(n=5)	86-104	95
UFH plasma(n=5)	90-104	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human SMAD2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	89-99%	87-104%	90-104%
EDTA plasma(n=5)	82-98%	82-100%	82-100%
UFH plasma(n=5)	81-95%	86-96%	80-91%

CV(%):

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Component	Quantity	Storage
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

Other materials and equipment required:

Microplate reader with 450 nm wavelength filter
Multichannel Pipette, Pipette, microcentrifuge tubes and disposable pipette tips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

Q15796

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:	SMAD2Â Â
NCBI Official Synonym Symbols:	JV18; MADH2; MADR2; JV18-1; hMAD-2; hSMAD2Â Â
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:	SMAD2Â Â
UniProt Entry Name:	SMAD2_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
1.	Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.	Aliquot 0.1ml standard solutions into the standard wells.
3.	Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.	Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.	Seal the plate with a cover and incubate at 37 °C for 90 min.
6.	Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.	Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.	Seal the plate with a cover and incubate at 37°C for 60 min.
9.	Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.	Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.	Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.	Add 90 Âµl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.	Add 50 Âµl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14.	Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

When carrying out an ELISA assay it is important to prepare your samples in order to achieve the best possible results. Below we have a list of procedures for the preparation of samples for different sample types.

Sample Type	Protocol
Serum	If using serum separator tubes, allow samples to clot for 30 minutes at room temperature. Centrifuge for 10 minutes at 1,000x g. Collect the serum fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. If serum separator tubes are not being used, allow samples to clot overnight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Remove serum and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles.
Plasma	Collect plasma using EDTA or heparin as an anticoagulant. Centrifuge samples at 4°C for 15 mins at 1000 × g within 30 mins of collection. Collect the plasma fraction and assay promptly or aliquot and store the samples at -80°C. Avoid multiple freeze-thaw cycles. Note: Over haemolysed samples are not suitable for use with this kit.
Urine & Cerebrospinal Fluid	Collect the urine (mid-stream) in a sterile container, centrifuge for 20 mins at 2000-3000 rpm. Remove supernatant and assay immediately. If any precipitation is detected, repeat the centrifugation step. A similar protocol can be used for cerebrospinal fluid.
Cell culture supernatant	Collect the cell culture media by pipette, followed by centrifugation at 4°C for 20 mins at 1500 rpm. Collect the clear supernatant and assay immediately.
Cell lysates	Solubilize cells in lysis buffer and allow to sit on ice for 30 minutes. Centrifuge tubes at 14,000 x g for 5 minutes to remove insoluble material. Aliquot the supernatant into a new tube and discard the remaining whole cell extract. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Tissue homogenates	The preparation of tissue homogenates will vary depending upon tissue type. Rinse tissue with 1X PBS to remove excess blood & homogenize in 20ml of 1X PBS (including protease inhibitors) and store overnight at ≤ -20°C. Two freeze-thaw cycles are required to break the cell membranes. To further disrupt the cell membranes you can sonicate the samples. Centrifuge homogenates for 5 mins at 5000xg. Remove the supernatant and assay immediately or aliquot and store at -20°C or -80°C.
Tissue lysates	Rinse tissue with PBS, cut into 1-2 mm pieces, and homogenize with a tissue homogenizer in PBS. Add an equal volume of RIPA buffer containing protease inhibitors and lyse tissues at room temperature for 30 minutes with gentle agitation. Centrifuge to remove debris. Quantify total protein concentration using a total protein assay. Assay immediately or aliquot and store at ≤ -20 °C.
Breast Milk	Collect milk samples and centrifuge at 10,000 x g for 60 min at 4°C. Aliquot the supernatant and assay. For long term use, store samples at -80°C. Minimize freeze/thaw cycles.