Product Name:	Mouse Beta Catenin/CTNNb1 ELISA Kit
Product Code:	MOFI00751
Size:	96 Assays
Alias:	CTNNb1, CTNNbeta1, beta-catenin, catenin, cadherin-associated protein, beta 1, 88kDa, CTNNB
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse CTNNb1 concentrations in serum plasma and other biological fluids.
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 Mouse CTNNb1 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse CTNNb1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-103	96
EDTA plasma(n=5)	88-105	97
UFH plasma(n=5)	89-104	97

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse CTNNb1 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)	85-95%	93-103%	89-103%
EDTA plasma(n=5)	87-101%	82-98%	82-97%
UFH plasma(n=5)	80-87%	87-100%	84-100%

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

Q02248

UniProt Protein Function:	CTNNB1: a regulator of cell adhesion and a key downstream effector in the Wnt signaling pathway. Implicated early embryonic development and tumorigenesis. Phosphorylated and destabilized by CK1 and GSK-3beta. Stabilized cytoplasmic beta-catenin is a hallmark of a variety of cancers. Stabilized beta-catenin translocates to the nucleus, where it acts as a transcriptional activator of T-cell factor (TCF)-regulated genes. Interacts with the PDZ domain of TAX1BP3, inhibiting its transcriptional activity. Two alternatively spliced human isoforms have been described.
UniProt Protein Details:	Protein type:Actin-binding; Cell adhesion; Transcription factor; Oncoprotein; Motility/polarity/chemotaxis; Nuclear receptor co-regulator Cellular Component: adherens junction; apical junction complex; apical part of cell; basolateral plasma membrane; beta-catenin destruction complex; catenin complex; cell cortex; cell junction; cell projection membrane; cell-cell adherens junction; centrosome; cytoplasm; cytoskeleton; cytosol; dendritic shaft; fascia adherens; flotillin complex; focal adhesion; intercellular junction; lamellipodium; lateral plasma membrane; membrane; microvillus membrane; neuron projection; nucleus; perinuclear region of cytoplasm; plasma membrane; protein complex; synapse; tight junction; transcription factor complex; Z disc Molecular Function:alpha-catenin binding; cadherin binding; chromatin binding; DNA binding; double-stranded DNA binding; enzyme binding; estrogen receptor binding; ionotropic glutamate receptor binding; kinase binding; nitric-oxide synthase binding; nuclear hormone receptor binding; protein binding; protein C-terminus binding; protein complex binding; protein kinase binding; protein phosphatase binding; signal transducer activity; SMAD binding; transcription coactivator activity; transcription factor activity; transcription factor binding Biological Process: anterior/posterior axis specification; bone resorption; cardiac muscle cell proliferation; cell adhesion; cell differentiation; cell fate determination; cell fate specification; cell maturation; cell proliferation; cell-cell adhesion; cell-matrix adhesion; cellular morphogenesis during differentiation; cellular process; central nervous system vasculogenesis; chromatin-mediated maintenance of transcription; dorsal/ventral axis specification; dorsal/ventral pattern formation; ectoderm development; embryonic axis specification; embryonic digit morphogenesis; embryonic foregut morphogenesis; embryonic forelimb morphogenesis; embryonic heart tube development; embryonic hindlimb morphogenesis; endoderm formation; endodermal cell fate commitment; fallopian tube development; forebrain development; gastrulation with mouth forming second; genitalia morphogenesis; glial cell fate determination; hair cycle process; hair follicle morphogenesis; heart development; hemopoiesis; hindbrain development; in utero embryonic development; kidney development; layer formation in the cerebral cortex; lens morphogenesis in camera-type eye; limb development; lung development; male genitalia development; midbrain development; morphogenesis of embryonic epithelium; negative regulation of cell differentiation; negative regulation of cell proliferation; negative regulation of chondrocyte differentiation; negative regulation of mitotic cell cycle, embryonic; negative regulation of oligodendrocyte differentiation; negative regulation of osteoclast differentiation; negative regulation of protein sumoylation; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; nervous system development; neural plate development; neuron differentiation; neuron migration; odontogenesis of dentine-containing teeth; oocyte development; organ development; osteoclast differentiation; pancreas development; patterning of blood vessels; positive regulation of apoptosis; positive regulation of cell proliferation; positive regulation of endothelial cell differentiation; positive regulation of epithelial cell differentiation; positive regulation of fibroblast growth factor receptor signaling pathway; positive regulation of I-kappaB kinase/NF-kappaB cascade; positive regulation of MAPKKK cascade; positive regulation of mesenchymal cell proliferation; positive regulation of neuroblast proliferation; positive regulation of neuron apoptosis; positive regulation of osteoblast differentiation; positive regulation of skeletal muscle development; positive regulation of telomerase activity; positive regulation of telomere maintenance via telomerase; positive regulation of transcription factor activity; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; protein heterooligomerization; proximal/distal pattern formation; regulation of apoptosis; regulation of cell differentiation; regulation of cell proliferation; regulation of centriole-centriole cohesion; regulation of epithelial cell differentiation; regulation of gene expression; regulation of histone H3-K4 methylation; regulation of myelination; regulation of osteoblast differentiation; regulation of osteoclast differentiation; regulation of smooth muscle cell proliferation; regulation of T cell proliferation; regulation of transcription from RNA polymerase II promoter; regulation of transcription, DNA-dependent; response to cytokine stimulus; response to estradiol stimulus; response to estrogen stimulus; Schwann cell proliferation; skeletal development; skin development; smooth muscle cell differentiation; stem cell maintenance; synapse organization and biogenesis; synaptic transmission; synaptic vesicle transport; T cell differentiation; T cell differentiation in the thymus; thymus development; transcription, DNA-dependent; ureteric bud branching; vasculature development; vasculogenesis; Wnt receptor signaling pathway; Wnt receptor signaling pathway through beta-catenin
NCBI Summary:	This gene encodes not only an important cytoplasmic component of the classical cadherin adhesion complex that forms the adherens junction in epithelia and mediates cell-cell adhesion in many other tissues but also a key signaling molecule in the canonical Wnt signaling pathway that controls cell growth and differentiation during both normal development and tumorigenesis. The gene product contains a central armadillo-repeat containing domain through which it binds the cytoplasmic tail of classical cadherins; meanwhile, it also binds alpha-catenin, which further links the cadherin complex to the actin cytoskeleton either directly or indirectly. Beta-catenin is therefore necessary for the adhesive function of classical cadherins. Another key function of this protein is to mediate the canonical Wnt signaling pathway and regulate gene transcription. Without Wnt signal, cytoplasmic beta-catenin that is not associated with the cadherin complex is quickly phosphorylated at the N-terminal Ser/Thr residues by the so called degradation complex containing axin, adenomatous polyposis coli (APC), casein kinase I, and GSK3B, then ubiquitylated by beta-TrCP, and degraded by the proteasome. However, in the presence of Wnt signal, the degradation complex is disrupted and the stabilized cytoplasmic beta-catenin translocates into the nucleus, where it binds various transcription factors and, together with these factors, regulates the transcription of many downstream genes. Mutations of this gene have been linked with various types of tumors. Alternatively spliced variants have been found for this gene. [provided by RefSeq, Sep 2009]
UniProt Code:	Q02248
NCBI GenInfo Identifier:	399310
NCBI Gene ID:	12387
NCBI Accession:	Q02248.1
UniProt Secondary Accession:	Q02248,Q922W1, Q9D335,
UniProt Related Accession:	Q02248
Molecular Weight:	85,471 Da
NCBI Full Name:	Catenin beta-1
NCBI Synonym Full Names:	catenin (cadherin associated protein), beta 1
NCBI Official Symbol:	Ctnnb1
NCBI Official Synonym Symbols:	Bfc; Mesc; Catnb
NCBI Protein Information:	catenin beta-1
UniProt Protein Name:	Catenin beta-1
UniProt Synonym Protein Names:	Beta-catenin
Protein Family:	Beta-catenin-like protein
UniProt Gene Name:	Ctnnb1
UniProt Entry Name:	CTNB1_MOUSE

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

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 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.