Rat Smad3/MADH3 ELISA Kit (RTFI01133)- High Sensitivity

Product Name:	Rat Smad3 (Mothers Against Decapentaplegic Homolog 3) ELISA Kit
Product Code:	RTFI01133
Size:	96 Assays
Target:	Rat Smad3
Alias:	Smad3, MADH3, SMAD family member 3, hMAD-3, hSMAD3, MAD homolog 3, mad protein homolog, MAD, mothers against decapentaplegic homolog 3, MAD, mothers against decapentaplegic homolog 3, Drosophila, mad3, Mad3, MADH3mad homolog JV15-2, mothers against decapentaplegic homolog 3, Mothers against DPP homolog 3
Reactivity:	Rat
Detection Method:	Sandwich ELISA, Double Antibody
Sensitivity:	0.094ng/ml
Range:	0.156-10ng/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Rat Smad3 and the recovery rates were calculated by comparing the measured value to the expected amount of Rat Smad3 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-99	92
EDTA plasma(n=5)	87-105	95
UFH plasma(n=5)	88-100	94

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Rat Smad3 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)	92-98%	88-102%	87-104%
EDTA plasma(n=5)	90-100%	82-90%	83-96%
UFH plasma(n=5)	81-100%	84-96%	81-95%

Intra-Assay:

CV <8%

Inter-Assay:

CV <10%

Uniprot:

P84025

UniProt Protein Function:	SMAD3: transcription factor phosphorylated and activated by TGF-beta-type receptors. A receptor-regulated Smad (R-smad). Binds directly to consensus DNA-binding elements in the promoters of target genes. In mouse required for establishemnt of the mucosal immune response and proper development of skeleton.
UniProt Protein Details:	Protein type:DNA-binding; Nuclear receptor co-regulator; Transcription factor Chromosomal Location of Human Ortholog: 8q24 Cellular Component: cytoplasm; nuclear chromatin; nuclear inner membrane; nucleoplasm; nucleus; plasma membrane; protein complex; receptor complex; transcription factor complex Molecular Function:beta-catenin binding; bHLH transcription factor binding; chromatin binding; chromatin DNA binding; collagen binding; DNA binding; double-stranded DNA binding; enzyme binding; identical protein binding; phosphatase binding; protein binding; protein heterodimerization activity; protein homodimerization activity; protein kinase binding; sequence-specific DNA binding; SMAD binding; transcription factor activity; transcription factor binding; transforming growth factor beta receptor binding; transforming growth factor beta receptor, pathway-specific cytoplasmic mediator activity; ubiquitin binding; ubiquitin protein ligase binding; zinc ion binding Biological Process: activin receptor signaling pathway; caspase activation; cell cycle arrest; developmental growth; embryonic cranial skeleton morphogenesis; embryonic foregut morphogenesis; embryonic pattern specification; endoderm development; evasion of host defenses by virus; gastrulation; heart looping; immune response; immune system development; in utero embryonic development; intercellular junction assembly and maintenance; liver development; mesoderm formation; negative regulation of apoptosis; negative regulation of cell growth; negative regulation of cell proliferation; negative regulation of fat cell differentiation; negative regulation of inflammatory response; negative regulation of mitotic cell cycle; negative regulation of osteoblast differentiation; negative regulation of osteoblast proliferation; negative regulation of protein catabolic process; negative regulation of transcription from RNA polymerase II promoter; osteoblast development; osteoblast differentiation; paraxial mesoderm morphogenesis; pericardium development; positive regulation of bone mineralization; positive regulation of cell migration; positive regulation of chondrocyte differentiation; positive regulation of focal adhesion formation; positive regulation of interleukin-1 beta production; positive regulation of nitric oxide biosynthetic process; positive regulation of positive chemotaxis; positive regulation of stress fiber formation; positive regulation of transcription factor import into nucleus; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of transforming growth factor-beta3 production; protein stabilization; regulation of binding; regulation of epithelial cell proliferation; regulation of immune response; regulation of striated muscle development; regulation of transcription from RNA polymerase II promoter; regulation of transforming growth factor beta receptor signaling pathway; regulation of transforming growth factor-beta2 production; response to hypoxia; skeletal development; SMAD protein complex assembly; somitogenesis; T cell activation; thyroid gland development; transforming growth factor beta receptor signaling pathway; transport; ureteric bud development; Wnt receptor signaling pathway through beta-catenin
NCBI Summary:	transcription factor; important component of the TGF-beta signaling pathway [RGD, Feb 2006]
UniProt Code:	P84025
NCBI GenInfo Identifier:	6981174
NCBI Gene ID:	25631
NCBI Accession:	NP_037227.1
UniProt Secondary Accession:	P84025,O09064, O09144, O14510, O35273, Q92940, Q93002 Q9GKR4,
UniProt Related Accession:	P84025
Molecular Weight:	48,081 Da
NCBI Full Name:	mothers against decapentaplegic homolog 3
NCBI Synonym Full Names:	SMAD family member 3
NCBI Official Symbol:	Smad3
NCBI Official Synonym Symbols:	mad3; Madh3; Smad 3
NCBI Protein Information:	mothers against decapentaplegic homolog 3
UniProt Protein Name:	Mothers against decapentaplegic homolog 3
UniProt Synonym Protein Names:	SMAD family member 3; SMAD 3; Smad3
Protein Family:	Mothers against decapentaplegic
UniProt Gene Name:	Smad3

Step	Procedure
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 µL 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 µL 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 clotovernight at 2-8°C. Centrifuge for 10 minutes at 1,000x g. Removeserum 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 anti-coagulant. 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 & homogenizein 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.