Mouse Notch-1 ELISA Kit (MOFI01239)- High Sensitivity

Product Name:	Mouse Notch-1 (Neurogenic locus notch homolog protein 1) ELISA Kit
Product Code:	MOFI01239
Size:	96 Assays
Alias:	Notch-1
Detection Method:	Sandwich ELISA
Application:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Notch-1 concentrations in serum plasma and other biological fluids.
Sensitivity:	4.688pg/ml
Range:	7.813-500pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:	Matrices listed below were spiked with certain level of Mouse Notch-1 and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Notch-1 in samples. Not Available.
Linearity:	The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Notch-1 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected. Not Available.
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

Q01705

UniProt Protein Function:	Notch 1: Functions as a receptor for membrane-bound ligands Jagged1, Jagged2 and Delta1 to regulate cell-fate determination. Upon ligand activation through the released notch intracellular domain (NICD) it forms a transcriptional activator complex with RBPJ/RBPSUH and activates genes of the enhancer of split locus. Affects the implementation of differentiation, proliferation and apoptotic programs. May be important for normal lymphocyte function. In altered form, may contribute to transformation or progression in some T-cell neoplasms. Involved in the maturation of both CD4+ and CD8+ cells in the thymus. May be important for follicular differentiation and possibly cell fate selection within the follicle. During cerebellar development, may function as a receptor for neuronal DNER and may be involved in the differentiation of Bergmann glia. Represses neuronal and myogenic differentiation. May enhance HIF1A function by sequestering HIF1AN away from HIF1A. Heterodimer of a C-terminal fragment N(TM) and an N- terminal fragment N(EC) which are probably linked by disulfide bonds. Interacts with DNER, DTX1, DTX2 and RBPJ/RBPSUH. Also interacts with MAML1, MAML2 and MAML3 which act as transcriptional coactivators for NOTCH1. The activated membrane-bound form interacts with AAK1 which promotes NOTCH1 stabilization. Forms a trimeric complex with FBXW7 and SGK1. Interacts with HIF1AN. HIF1AN negatively regulates the function of notch intracellular domain (NICD), accelerating myogenic differentiation. In fetal tissues most abundant in spleen, brain stem and lung. Also present in most adult tissues where it is found mainly in lymphoid tissues. Belongs to the NOTCH family.
UniProt Protein Details:	Protein type:Membrane protein, integral; Motility/polarity/chemotaxis; Oncoprotein; Receptor, misc.; Transcription factor Chromosomal Location of Human Ortholog: 2 A3\|2 18.91 cM Cellular Component: acrosome; adherens junction; apical plasma membrane; cell surface; cytoplasm; cytoplasmic vesicle; endoplasmic reticulum; Golgi apparatus; integral to plasma membrane; nucleus; plasma membrane; receptor complex Molecular Function:chromatin binding; chromatin DNA binding; enzyme binding; enzyme inhibitor activity; Notch binding; protein binding; protein heterodimerization activity; sequence-specific DNA binding; transcription factor activity Biological Process: activation of Notch receptor target transcription factor; anagen; astrocyte differentiation; auditory receptor cell fate commitment; axonogenesis; branching morphogenesis of a tube; cardiac muscle cell proliferation; cardiac muscle morphogensis; cell differentiation; cell fate commitment; cell fate specification; compartment specification; determination of left/right symmetry; embryonic hindlimb morphogenesis; embryonic limb morphogenesis; endoderm development; epidermis development; epithelial to mesenchymal transition; forebrain development; foregut morphogenesis; glial cell differentiation; hair follicle morphogenesis; heart development; heart looping; humoral immune response; in utero embryonic development; inflammatory response to antigenic stimulus; keratinocyte differentiation; liver development; lumen formation; lung development; mesenchymal cell development; negative regulation of auditory receptor cell differentiation; negative regulation of BMP signaling pathway; negative regulation of calcium ion-dependent exocytosis; negative regulation of catalytic activity; negative regulation of cell differentiation; negative regulation of cell proliferation; negative regulation of myoblast differentiation; negative regulation of neurogenesis; negative regulation of neuron differentiation; negative regulation of oligodendrocyte differentiation; negative regulation of ossification; negative regulation of osteoblast differentiation; negative regulation of photoreceptor cell differentiation; negative regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; neural tube development; neuron differentiation; neuron fate commitment; Notch signaling pathway; positive regulation of apoptosis; positive regulation of astrocyte differentiation; positive regulation of BMP signaling pathway; positive regulation of cardiac muscle cell proliferation; positive regulation of cell migration; positive regulation of cell proliferation; positive regulation of endothelial cell differentiation; positive regulation of epithelial cell proliferation; positive regulation of glial cell differentiation; positive regulation of JAK-STAT cascade; positive regulation of keratinocyte differentiation; positive regulation of neuroblast proliferation; positive regulation of Notch signaling pathway; positive regulation of transcription from RNA polymerase II promoter; positive regulation of transcription, DNA-dependent; positive regulation of viral genome replication; regulation of auditory receptor cell differentiation; regulation of cell migration; regulation of cell proliferation; regulation of epithelial cell proliferation; regulation of gene expression; regulation of neurogenesis; regulation of Notch signaling pathway; regulation of somitogenesis; regulation of transcription from RNA polymerase II promoter; response to muramyl dipeptide; somatic stem cell division; spermatogenesis; sprouting angiogenesis; venous blood vessel morphogenesis
UniProt Code:	Q01705
NCBI GenInfo Identifier:	384872684
NCBI Gene ID:	18128
NCBI Accession:	Q01705.3
UniProt Secondary Accession:	Q01705,Q06007, Q3TZW2, Q3U3Y2, Q61905, Q7TQ50, Q7TQ51 Q7TQ52, Q8K428, Q99JC2, Q9QW58, Q9R0X7,
UniProt Related Accession:	Q01705
Molecular Weight:	269,180 Da
NCBI Full Name:	Neurogenic locus notch homolog protein 1
NCBI Synonym Full Names:	notch 1
NCBI Official Symbol:	Notch1
NCBI Official Synonym Symbols:	N1; Mis6; Tan1; lin-12; 9930111A19Rik
NCBI Protein Information:	neurogenic locus notch homolog protein 1
UniProt Protein Name:	Neurogenic locus notch homolog protein 1
UniProt Synonym Protein Names:	Motch A; mT14; p300
Protein Family:	Neurogenic locus notch homolog protein
UniProt Gene Name:	Notch1

*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.