Human NOTCH1 ELISA Kit (HUFI00699)- High Sensitivity

Product Name:	Human NOTCH1 ELISA Kit
Product Code:	HUFI00699
Size:	96 Assays
Alias:	NOTCH1, hN1, Neurogenic locus notch homolog protein 1, TAN1, Translocation-associated notch protein TAN-1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human NOTCH1 concentrations in serum plasma and other biological fluids.
Sensitivity:	46.875pg/ml
Range:	78.125-5000pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human NOTCH1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human NOTCH1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-104	93
EDTA plasma(n=5)	91-102	98
UFH plasma(n=5)	90-101	96

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human NOTCH1 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)	88-105%	88-96%	85-105%
EDTA plasma(n=5)	82-101%	89-99%	86-99%
UFH plasma(n=5)	87-99%	83-98%	88-100%

CV(%):

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

P46531

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:Motility/polarity/chemotaxis; Membrane protein, integral; Oncoprotein; Transcription factor; Receptor, misc. Chromosomal Location of Human Ortholog: 9q34.3 Cellular Component: nucleoplasm; Golgi membrane; endoplasmic reticulum membrane; cell surface; integral to membrane; acrosome; extracellular region; plasma membrane; cytosol; nucleus; receptor complex Molecular Function:enzyme inhibitor activity; protein binding; enzyme binding; chromatin DNA binding; sequence-specific DNA binding; receptor activity; calcium ion binding; transcription factor activity Biological Process: neural tube development; negative regulation of calcium ion-dependent exocytosis; positive regulation of apoptosis; heart development; positive regulation of transcription, DNA-dependent; positive regulation of JAK-STAT cascade; response to lipopolysaccharide; cell differentiation in spinal cord; negative regulation of BMP signaling pathway; response to corticosteroid stimulus; compartment specification; positive regulation of endothelial cell differentiation; negative regulation of ossification; oligodendrocyte differentiation; somatic stem cell division; negative regulation of osteoblast differentiation; positive regulation of astrocyte differentiation; positive regulation of cardiac muscle cell proliferation; positive regulation of keratinocyte differentiation; negative regulation of photoreceptor cell differentiation; positive regulation of neuroblast proliferation; organ regeneration; Notch receptor processing; keratinocyte differentiation; branching morphogenesis of a tube; response to muramyl dipeptide; positive regulation of transcription from RNA polymerase II promoter; negative regulation of transcription, DNA-dependent; determination of left/right symmetry; positive regulation of epithelial cell proliferation; foregut morphogenesis; endoderm development; cardiac muscle cell proliferation; cell fate specification; negative regulation of transcription from RNA polymerase II promoter; embryonic hindlimb morphogenesis; negative regulation of neurogenesis; negative regulation of cell proliferation; astrocyte differentiation; regulation of transcription, DNA-dependent; tissue regeneration; cardiac muscle morphogensis; positive regulation of cell proliferation; forebrain development; heart looping; regulation of somitogenesis; positive regulation of BMP signaling pathway; mesenchymal cell development; transcription initiation from RNA polymerase II promoter; Notch signaling pathway; hair follicle morphogenesis; in utero embryonic development; lumen formation; liver development; humoral immune response; activation of Notch receptor target transcription factor; negative regulation of oligodendrocyte differentiation; inflammatory response to antigenic stimulus; axonogenesis; negative regulation of catalytic activity; epithelial to mesenchymal transition; spermatogenesis; gene expression; immune response; sprouting angiogenesis; negative regulation of myoblast differentiation; auditory receptor cell fate commitment; positive regulation of cell migration; lung development Disease: Adams-oliver Syndrome 5; Aortic Valve Disease 1
NCBI Summary:	This gene encodes a member of the Notch family. Members of this Type 1 transmembrane protein family share structural characteristics including an extracellular domain consisting of multiple epidermal growth factor-like (EGF) repeats, and an intracellular domain consisting of multiple, different domain types. Notch family members play a role in a variety of developmental processes by controlling cell fate decisions. The Notch signaling network is an evolutionarily conserved intercellular signaling pathway which regulates interactions between physically adjacent cells. In Drosophilia, notch interaction with its cell-bound ligands (delta, serrate) establishes an intercellular signaling pathway that plays a key role in development. Homologues of the notch-ligands have also been identified in human, but precise interactions between these ligands and the human notch homologues remain to be determined. This protein is cleaved in the trans-Golgi network, and presented on the cell surface as a heterodimer. This protein functions as a receptor for membrane bound ligands, and may play multiple roles during development. [provided by RefSeq]
UniProt Code:	P46531
NCBI GenInfo Identifier:	11275980
NCBI Gene ID:	4851
NCBI Accession:
UniProt Secondary Accession:	P46531,Q59ED8, Q5SXM3,
UniProt Related Accession:	P46531,Q6IAD4,Q9NXX4
Molecular Weight:	272,505 Da
NCBI Full Name:	NOTCH 1
NCBI Synonym Full Names:	notch 1
NCBI Official Symbol:	NOTCH1
NCBI Official Synonym Symbols:	hN1; TAN1
NCBI Protein Information:	neurogenic locus notch homolog protein 1; OTTHUMP00000022594; Notch homolog 1, translocation-associated; translocation-associated notch protein TAN-1
UniProt Protein Name:	Neurogenic locus notch homolog protein 1
UniProt Synonym Protein Names:	Translocation-associated notch protein TAN-1
Protein Family:	Neurogenic locus notch homolog protein
UniProt Gene Name:	NOTCH1
UniProt Entry Name:	NOTC1_HUMAN

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

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37°C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Step	Protocol
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 Âµl 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 Âµl 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.

Human NOTCH1 ELISA Kit (HUFI00699)

Description