Human NOTCH1 ELISA Kit (HUEB0692)- High Sensitivity

Product Name:	Human Neurogenic locus notch homolog protein 1 (NOTCH1) ELISA Kit
SKU:	HUEB0692
Size:	96T
Target:	Human Neurogenic locus notch homolog protein 1 (NOTCH1)
Synonyms:	Translocation-associated notch protein TAN-1, Notch 1, TAN1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	78-5000pg/mL
Sensitivity:	49pg/mL

Intra CV:

5.6%

Inter CV:

10.2%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	99-109%	110-120%	87-95%	94-106%
EDTA Plasma(N=5)	88-98%	101-113%	102-111%	117-127%
Heparin Plasma(N=5)	112-122%	90-100%	104-117%	89-98%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	102	96-108
Plasma	104	98-110

Function:

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. Involved in angiogenesis; negatively regulates endothelial cell proliferation and migration and angiogenic sprouting. Involved in the maturation of both CD4+ and CD8+ cells in the thymus. Important for follicular differentiation and possibly cell fate selection within the follicle. During cerebellar development, functions as a receptor for neuronal DNER and is involved in the differentiation of Bergmann glia. Represses neuronal and myogenic differentiation. May play an essential role in postimplantation development, probably in some aspect of cell specification and/or differentiation. May be involved in mesoderm development, somite formation and neurogenesis. May enhance HIF1A function by sequestering HIF1AN away from HIF1A. Required for the THBS4 function in regulating protective astrogenesis from the subventricular zone (SVZ) niche after injury. Involved in determination of left/right symmetry by modulating the balance between motile and immotile (sensory) cilia at the left-right organiser (LRO).

Uniprot:	P46531
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Neurogenic locus notch homolog protein 1
Sub Unit:	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 (PubMed:11101851, PubMed:12370315). Notch 1 intracellular domain interacts with SNW1; the interaction involves multimerized NOTCH1 NICD and is implicated in a formation of an intermediate preactivation complex which associates with DNA-bound CBF-1/RBPJ (PubMed:10713164). 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 (PubMed:17573339). Interacts (via NICD) with SNAI1 (via zinc fingers); the interaction induces SNAI1 degradation via MDM2-mediated ubiquitination and inhibits SNAI1-induced cell invasion. Interacts (via NICD) with MDM2A. Interacts (via NICD) with BCL6; the interaction decreases MAML1 recruitment by NOTCH1 NICD on target genes DNA and inhibits NOTCH1 transcractivation activity. Interacts with THBS4 (By similarity). Interacts (via the EGF-like repeat region) with NOV (via CTCK domain) (PubMed:12050162). Interacts (via EGF-like domains) with DLL4 (via N-terminal DSL and MNNL domains) (By similarity). Interacts with ZMIZ1.
Research Area:	Neurosciences
Subcellular Location:	Notch 1 intracellular domain Nucleus Following proteolytical processing NICD is translocated to the nucleus.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

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

Component	Quantity (96 Assays)	Storage
ELISA Microplate (Dismountable)	8×12 strips	-20°C
Lyophilized Standard	2	-20°C
Sample Diluent	20ml	-20°C
Assay Diluent A	10mL	-20°C
Assay Diluent B	10mL	-20°C
Detection Reagent A	120µL	-20°C
Detection Reagent B	120µL	-20°C
Wash Buffer	30mL	4°C
Substrate	10mL	4°C
Stop Solution	10mL	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

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

Allow all reagents to reach room temperature (Please do not dissolve the reagents at 37°C directly). All the reagents should be mixed thoroughly by gently swirling before pipetting. Avoid foaming. Keep appropriate numbers of strips for 1 experiment and remove extra strips from microtiter plate. Removed strips should be resealed and stored at -20°C until the kits expiry date. Prepare all reagents, working standards and samples as directed in the previous sections. Please predict the concentration before assaying. If values for these are not within the range of the standard curve, users must determine the optimal sample dilutions for their experiments. We recommend running all samples in duplicate.

Step
1.	Add Sample: Add 100µL of Standard, Blank, or Sample per well. The blank well is added with Sample diluent. Solutions are added to the bottom of micro ELISA plate well, avoid inside wall touching and foaming as possible. Mix it gently. Cover the plate with sealer we provided. Incubate for 120 minutes at 37°C.
2.	Remove the liquid from each well, don't wash. Add 100µL of Detection Reagent A working solution to each well. Cover with the Plate sealer. Gently tap the plate to ensure thorough mixing. Incubate for 1 hour at 37°C. Note: if Detection Reagent A appears cloudy warm to room temperature until solution is uniform.
3.	Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 400µL) (a squirt bottle, multi-channel pipette,manifold dispenser or automated washer are needed). Complete removal of liquid at each step is essential. After the last wash, completely remove remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	Add 100µL of Detection Reagent B working solution to each well. Cover with the Plate sealer. Incubate for 60 minutes at 37°C.
5.	Repeat the wash process for five times as conducted in step 3.
6.	Add 90µL of Substrate Solution to each well. Cover with a new Plate sealer and incubate for 10-20 minutes at 37°C. Protect the plate from light. The reaction time can be shortened or extended according to the actual color change, but this should not exceed more than 30 minutes. When apparent gradient appears in standard wells, user should terminatethe reaction.
7.	Add 50µL of Stop Solution to each well. If color change does not appear uniform, gently tap the plate to ensure thorough mixing.
8.	Determine the optical density (OD value) of each well at once, using a micro-plate reader set to 450 nm. User should open the micro-plate reader in advance, preheat the instrument, and set the testing parameters.
9.	After experiment, store all reagents according to the specified storage temperature respectively until their expiry.

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.

ELISA

Human NOTCH1 ELISA Kit