Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Human
Detection method:	Chemiluminescence
Detection range:	0.16-10 ng/mL
Sensitivity:	0.09 ng/mL
Sample volume:	100µL
Sample type:	Serum, plasma and other biological fluids
Repeatability:	CV < 15%
Specificity:	This kit recognizes Human Smad1/MADH1 in samples. No significant cross-reactivity or interference between Human Smad1/MADH1 and analogues was observed.

This kit uses Sandwich-CLIA as the method. The micro CLIA plate provided in this kit has been pre-coated with an antibody specific to Human Smad1/MADH1. Standards or samples are added to the appropriate micro CLIA plate wells and combined with the specific antibody. Then a biotinylated detection antibody specific for Human Smad1/MADH1 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 Smad1/MADH1, biotinylated detection antibody and Avidin-HRP conjugate will appear fluorescence. The Relative light unit (RLU) value is measured spectrophotometrically by the Chemiluminescence immunoassay analyzer. The RLU value is positively associated with the concentration of Human Smad1/MADH1. The concentration of Human Smad1/MADH1 in the samples can be calculated by comparing the RLU of the samples to the standard curve.

UniProt Protein Function:	SMAD1: transcription factor phosphorylated and activated by bone morphogenetic protein type 1 receptor kinases. Participates in a wide range of critical processes including morphogenesis, cell-fate determination, proliferation, differentiation and apoptosis. Phosphorylated forms dimerize with collaborating Smad4 and are translocated into the nucleus, where the transcription of target genes is stimulated.
UniProt Protein Details:	Protein type:DNA-binding; Transcription factor Chromosomal Location of Human Ortholog: 4q31 Cellular Component: nucleoplasm; transcription factor complex; protein complex; cytoplasm; integral to membrane; nuclear inner membrane; intracellular; cytosol; nucleus Molecular Function:identical protein binding; protein binding; metal ion binding; receptor signaling protein activity; protein kinase binding; transcription factor activity; transforming growth factor beta receptor, pathway-specific cytoplasmic mediator activity Biological Process: wound healing; cardiac muscle cell proliferation; primary microRNA processing; embryonic pattern specification; signal transduction; protein amino acid phosphorylation; mesodermal cell fate commitment; BMP signaling pathway; negative regulation of cell proliferation; homeostatic process; ureteric bud development; transforming growth factor beta receptor signaling pathway; midbrain development; inflammatory response; hindbrain development; response to drug; transcription, DNA-dependent; MAPKKK cascade; positive regulation of dendrite morphogenesis; response to organic nitrogen; SMAD protein complex assembly; positive regulation of osteoblast differentiation; gamete generation; cartilage development; positive regulation of transcription from RNA polymerase II promoter; osteoblast fate commitment
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 signals of the bone morphogenetic proteins (BMPs), which are involved in a range of biological activities including cell growth, apoptosis, morphogenesis, development and immune responses. In response to BMP ligands, this protein can be phosphorylated and activated by the BMP receptor kinase. The phosphorylated form of this protein forms a complex with SMAD4, which is important for its function in the transcription regulation. This protein is a target for SMAD-specific E3 ubiquitin ligases, such as SMURF1 and SMURF2, and undergoes ubiquitination and proteasome-mediated degradation. Alternatively spliced transcript variants encoding the same protein have been observed. [provided by RefSeq, Jul 2008]
UniProt Code:	Q15797
NCBI GenInfo Identifier:	13633915
NCBI Gene ID:	4086
NCBI Accession:	Q15797. 1
UniProt Secondary Accession:	Q15797,Q16636, Q9UFT8, A8KAJ0, D3DNZ9,
UniProt Related Accession:	Q15797
Molecular Weight:	465
NCBI Full Name:	Mothers against decapentaplegic homolog 1
NCBI Synonym Full Names:	SMAD family member 1
NCBI Official Symbol:	SMAD1
NCBI Official Synonym Symbols:	BSP1; JV41; BSP-1; JV4-1; MADH1; MADR1
NCBI Protein Information:	mothers against decapentaplegic homolog 1; MAD homolog 1; Mad-related protein 1; TGF-beta signaling protein 1; mothers against DPP homolog 1; SMAD, mothers against DPP homolog 1; MAD, mothers against decapentaplegic homolog 1; transforming growth factor-beta signaling protein 1; transforming growth factor-beta-signaling protein 1
UniProt Protein Name:	Mothers against decapentaplegic homolog 1
UniProt Synonym Protein Names:	JV4-1; Mad-related protein 1; SMAD family member 1; SMAD 1; Smad1; hSMAD1; Transforming growth factor-beta-signaling protein 1; BSP-1
Protein Family:	Mothers against decapentaplegic
UniProt Gene Name:	SMAD1
UniProt Entry Name:	SMAD1_HUMAN

As the RLU 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.

Concentration (ng/mL)	RLU	Average	Corrected
10	30095 34847	32471	32447
5	14498 16442	15470	15446
2.5	7565 7441	7503	7479
1.25	3425 3881	3653	3629
0.63	1820 1702	1761	1737
0.31	839 809	824	800
0.16	350 364	357	333
0	23 25	24	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Human Smad1/MADH1 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Human Smad1/MADH1 were tested on 3 different plates, 20 replicates in each plate.

	Intra-assay Precision			Inter-assay Precision
Sample	1	2	3	1	2	3
n	20	20	20	20	20	20
Mean (ng/mL)	0.51	1.02	4.12	0.49	0.97	4.49
Standard deviation	0.06	0.11	0.30	0.06	0.10	0.29
C V (%)	11.76	10.78	7.28	12.24	10.31	6.46

Recovery

The recovery of Human Smad1/MADH1 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)	88-102	93
EDTA plasma (n=5)	100-112	106
Cell culture media (n=5)	94-111	102

Linearity

Samples were spiked with high concentrations of Human Smad1/MADH1 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)
1:2	Range (%)	85-97	86-98	93-108
1:2	Average (%)	92	92	99
1:4	Range (%)	95-109	100-118	89-103
1:4	Average (%)	103	108	94
1:8	Range (%)	87-98	94-110	96-110
1:8	Average (%)	92	101	102
1:16	Range (%)	92-108	91-104	91-108
1:16	Average (%)	99	97	98

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 CLIA Plate(Dismountable)	8 wells ×12 strips	-20°C, 6 months
Reference Standard	2 vials
Concentrated Biotinylated Detection Ab (100×)	1 vial, 120 µL
Concentrated HRP Conjugate (100×)	1 vial, 120 µL	-20°C(shading light), 6 months
Reference Standard & Sample Diluent	1 vial, 20 mL	4°C, 6 months
Biotinylated Detection Ab Diluent	1 vial, 14 mL
HRP Conjugate Diluent	1 vial, 14 mL
Concentrated Wash Buffer (25×)	1 vial, 30 mL
Substrate Reagent A	1 vial, 5 mL	4°C (shading light)
Substrate Reagent B	1 vial, 5 mL	4°C (shading light)
Plate Sealer	5 pieces
Product Description	1 copy
Certificate of Analysis	1 copy

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.
Aliquot 100µl of standard solutions into the standard wells.
Add 100µl of Sample / Standard dilution buffer into the control (zero) well.
Add 100µl of properly diluted sample (serum, plasma, tissue homogenates and otherbiological fluids. ) into test sample wells.
Cover the plate with the sealer provided in the kit and incubate for 90 min at 37°C.
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.
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.
Add 100µL of HRP Conjugate working solution to each well. Cover with a plate seal andincubate for 30 min at 37°C.
Aspirate or decant the solution from each well. Repeat the wash process for five times asconducted in step 7.
Add 100µL of Substrate mixture solution to each well. Cover with a new plate seal andincubate for no more than 5 min at 37°C. Protect the plate from light.
Determine the RLU value of each well immediately.