Assay type:	Sandwich
Format:	96T
Assay time:	4.5h
Reactivity:	Mouse
Detection Method:	Colormetric
Detection Range:	0.31-20 ng/mL
Sensitivity:	0.19 ng/mL
Sample Volume Required Per Well:	100µL
Sample Type:	Serum, plasma and other biological fluids
Specificity:	This kit recognizes Mouse RUNX2 in samples. No significant cross-reactivity or interference between Mouse RUNX2 and analogues was observed.

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 Mouse RUNX2. 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 Mouse RUNX2 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 Mouse RUNX2, 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 Mouse RUNX2. The concentration of Mouse RUNX2 in samples can be calculated by comparing the OD of the samples to the standard curve.

UniProt Protein Function:	AML3: Transcription factor involved in osteoblastic differentiation and skeletal morphogenesis. Essential for the maturation of osteoblasts and both intramembranous and endochondral ossification. CBF binds to the core site, 5'- PYGPYGGT-3', of a number of enhancers and promoters, including murine leukemia virus, polyomavirus enhancer, T-cell receptor enhancers, osteocalcin, osteopontin, bone sialoprotein, alpha 1(I) collagen, LCK, IL-3 and GM-CSF promoters. In osteoblasts, supports transcription activation: synergizes with SPEN/MINT to enhance FGFR2-mediated activation of the osteocalcin FGF-responsive element (OCFRE). Inhibits KAT6B-dependent transcriptional activation. Interaction with SATB2 results in enhanced DNA binding and transactivation by these transcription factors. Heterodimer of an alpha and a beta subunit. Interacts with HIVEP3 and HIPK3. The alpha subunit binds DNA as a monomer and through the Runt domain. DNA-binding is increased by heterodimerization. Interacts with XRCC6 (Ku70) and XRCC5 (Ku80). Interacts with KAT6A and KAT6B. Binds to cyclin B1 CCNB1. Interacts with DDX5. Specifically expressed in osteoblasts. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Transcription factor; DNA-binding Chromosomal Location of Human Ortholog: 6p21 Cellular Component: nucleoplasm; transcription factor complex; cytoplasm; nuclear chromatin Molecular Function:protein domain specific binding; protein binding; bHLH transcription factor binding; chromatin binding; transcription factor activity; ATP binding Biological Process: embryonic forelimb morphogenesis; transcription initiation from RNA polymerase II promoter; ossification; positive regulation of transcription, DNA-dependent; cell maturation; regulation of fibroblast growth factor receptor signaling pathway; chondrocyte development; embryonic cranial skeleton morphogenesis; stem cell differentiation; osteoblast development; odontogenesis of dentine-containing teeth; BMP signaling pathway; osteoblast differentiation; positive regulation of osteoblast differentiation; positive regulation of chondrocyte differentiation; negative regulation of smoothened signaling pathway; positive regulation of cell proliferation; gene expression; negative regulation of transcription, DNA-dependent; regulation of odontogenesis of dentine-containing teeth; osteoblast fate commitment; T cell differentiation; endochondral ossification Disease: Metaphyseal Dysplasia With Maxillary Hypoplasia With Or Without Brachydactyly; Cleidocranial Dysplasia
NCBI Summary:	This gene is a member of the RUNX family of transcription factors and encodes a nuclear protein with an Runt DNA-binding domain. This protein is essential for osteoblastic differentiation and skeletal morphogenesis and acts as a scaffold for nucleic acids and regulatory factors involved in skeletal gene expression. The protein can bind DNA both as a monomer or, with more affinity, as a subunit of a heterodimeric complex. Two regions of potential trinucleotide repeat expansions are present in the N-terminal region of the encoded protein, and these and other mutations in this gene have been associated with the bone development disorder cleidocranial dysplasia (CCD). Transcript variants that encode different protein isoforms result from the use of alternate promoters as well as alternate splicing. [provided by RefSeq, Jul 2016]
UniProt Code:	Q08775
NCBI GenInfo Identifier:	17368460
NCBI Gene ID:	860
NCBI Accession:	Q13950. 2
UniProt Secondary Accession:	Q08775,Q08775, Q9Z2J9,
UniProt Related Accession:	Q13950
Molecular Weight:
NCBI Full Name:	Runt-related transcription factor 2
NCBI Synonym Full Names:	RUNX family transcription factor 2
NCBI Official Symbol:	RUNX2
NCBI Official Synonym Symbols:	CCD; AML3; CCD1; CLCD; OSF2; CBFA1; OSF-2; PEA2aA; PEBP2aA; CBF-alpha-1
NCBI Protein Information:	runt-related transcription factor 2
UniProt Protein Name:	Runt-related transcription factor 2
UniProt Synonym Protein Names:	Acute myeloid leukemia 3 protein; Core-binding factor subunit alpha-1; CBF-alpha-1; Oncogene AML-3; Osteoblast-specific transcription factor 2; OSF-2; Polyomavirus enhancer-binding protein 2 alpha A subunit; PEA2-alpha A; PEBP2-alpha A; SL3-3 enhancer factor 1 alpha A subunit; SL3/AKV core-binding factor alpha A subunit
Protein Family:	Runt-related transcription factor
UniProt Gene Name:	RUNX2
UniProt Entry Name:	RUNX2_HUMAN

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.

Concentration (ng/mL)	O.D	Average	Corrected
20	2.345 2.393	2.369	2.283
10	1.493 1.515	1.504	1.418
5	0.907 0.897	0.902	0.816
2.5	0.406 0.42	0.413	0.327
1.25	0.244 0.226	0.235	0.149
0.63	0.185 0.171	0.178	0.092
0.31	0.129 0.137	0.133	0.047
0	0.081 0.091	0.086	--

Precision

Intra-assay Precision (Precision within an assay): 3 samples with low, mid range and high level Mouse RUNX2 were tested 20 times on one plate, respectively.

Inter-assay Precision (Precision between assays): 3 samples with low, mid range and high level Mouse RUNX2 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)	1.00	2.78	8.30	1.01	2.97	8.50
Standard deviation	0.06	0.16	0.41	0.06	0.16	0.40
C V (%)	6.00	5.76	4.94	5.94	5.39	4.71

Recovery

The recovery of Mouse RUNX2 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)	87-100	93
EDTA plasma (n=5)	91-104	98
Cell culture media (n=5)	85-97	92

Linearity

Samples were spiked with high concentrations of Mouse RUNX2 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 (%)	98-114	91-107	90-105
1:2	Average (%)	105	99	96
1:4	Range (%)	89-103	85-96	89-102
1:4	Average (%)	95	90	94
1:8	Range (%)	87-100	84-98	87-102
1:8	Average (%)	94	91	93
1:16	Range (%)	95-107	85-99	83-95
1:16	Average (%)	100	92	87

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 ×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	1 vial, 10 mL	4°C(shading light)
Stop Solution	1 vial, 10 mL	4°C
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 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.
Add 50 µL of Stop Solution to each well. Note: Adding the stop solution should be done inthe same order as the substrate solution.
Determine the optical density (OD value) of each well immediately with a microplate readerset at 450 nm.