Rat Neurogenic locus notch homolog protein 1 (Notch1) ELISA Kit (RTEB0309)

Product Name:	Rat Neurogenic locus notch homolog protein 1 (Notch1) ELISA Kit
SKU:	RTEB0309
Size:	96T
Target:	Rat Neurogenic locus notch homolog protein 1 (Notch1)
Synonyms:	Notch 1
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	78-5000pg/mL
Sensitivity:	39.4pg/mL

Intra CV:

7.8%

Inter CV:

9.9%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	104-113%	104-114%	104-113%	93-104%
EDTA Plasma(N=5)	105-114%	87-96%	84-94%	113-125%
Heparin Plasma(N=5)	98-111%	106-118%	98-107%	98-108%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	100	94-106
Plasma	102	96-108

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:	Q07008
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat 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. 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. 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. 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 (By similarity). Interacts with THBS4 (By similarity). Interacts (via the EGF-like repeat region) with NOV (via CTCK domain) (By similarity). Interacts (via EGF-like domains) with DLL4 (via N-terminal DSL and MNNL domains) (PubMed:25700513). 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.Protein type: Membrane protein, integral; Transcription factor; Receptor, misc.; Oncoprotein; Motility/polarity/chemotaxisCellular Component: acrosome; adherens junction; apical plasma membrane; cell surface; cytoplasm; cytoplasmic vesicle; cytoskeleton; endoplasmic reticulum; Golgi apparatus; integral to membrane; lamellipodium; nucleus; plasma membrane; receptor complex; ruffleMolecular Function: calcium ion binding; chromatin binding; chromatin DNA binding; enzyme binding; enzyme inhibitor activity; Notch binding; protein heterodimerization activity; receptor activity; sequence-specific DNA binding; transcription factor activityBiological Process: activation of Notch receptor target transcription factor; astrocyte differentiation; auditory receptor cell fate commitment; axonogenesis; brain development; branching morphogenesis of a tube; cardiac muscle cell proliferation; cardiac muscle morphogensis; cell differentiation; cell differentiation in spinal cord; 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; oligodendrocyte differentiation; organ regeneration; 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; positive regulation of viral transcription; 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 corticosteroid stimulus; response to lipopolysaccharide; response to muramyl dipeptide; somatic stem cell division; spermatogenesis; sprouting angiogenesis; tissue regeneration; transcription from RNA polymerase II promoter
UniProt Protein Details:
NCBI Summary:	transmembrane receptor; involved in cell-cell interactions important for development and pattern formation [RGD, Feb 2006]
UniProt Code:	Q07008
NCBI GenInfo Identifier:	157787099
NCBI Gene ID:	25496
NCBI Accession:	NP_001099191.1
UniProt Secondary Accession:	Q07008,F1M9E7
UniProt Related Accession:	Q07008
Molecular Weight:	270,822 Da
NCBI Full Name:	neurogenic locus notch homolog protein 1
NCBI Synonym Full Names:	notch 1
NCBI Official Symbol:	Notch1
NCBI Official Synonym Symbols:	TAN1; NOTCH
NCBI Protein Information:	neurogenic locus notch homolog protein 1
UniProt Protein Name:	Neurogenic locus notch homolog protein 1
UniProt Synonym Protein Names:
Protein Family:	Neurogenic locus notch homolog protein
UniProt Gene Name:	Notch1
UniProt Entry Name:	NOTC1_RAT

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.