Human NOTCH4 ELISA Kit (HUEB2353)- High Sensitivity

Product Name:	Human Neurogenic locus notch homolog protein 4 (NOTCH4) ELISA Kit
SKU:	HUEB2353
Size:	96T
Target:	Human Neurogenic locus notch homolog protein 4 (NOTCH4)
Synonyms:	Notch 4, INT3
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Human
Detection Range:	0.312-20ng/mL
Sensitivity:	0.1ng/mL

Intra CV:

3.4%

Inter CV:

7.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	110-120%	99-109%	86-99%	96-106%
EDTA Plasma(N=5)	102-112%	92-100%	80-92%	104-114%
Heparin Plasma(N=5)	90-100%	88-98%	104-114%	107-117%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	97	91-103
Plasma	99	93-105

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. May regulate branching morphogenesis in the developing vascular system.

Uniprot:	Q99466
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant human Neurogenic locus notch homolog protein 4
Sub Unit:	Heterodimer of a C-terminal fragment N(TM) and a N-terminal fragment N(EC) which are probably linked by disulfide bonds (By similarity). Interacts with MAML1, MAML2 and MAML3 which act as transcriptional coactivators for NOTCH4.
Research Area:	Development Biology
Subcellular Location:	Notch 4 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:	NOTCH4: 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 regulate branching morphogenesis in the developing vascular system. Heterodimer of a C-terminal fragment N(TM) and a N- terminal fragment N(EC) which are probably linked by disulfide bonds. Interacts with MAML1, MAML2 and MAML3 which act as transcriptional coactivators for NOTCH4. Highly expressed in the heart, moderately in the lung and placenta and at low levels in the liver, skeletal muscle, kidney, pancreas, spleen, lymph node, thymus, bone marrow and fetal liver. No expression was seen in adult brain or peripheral blood leukocytes. Belongs to the NOTCH family. 3 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Membrane protein, integral Chromosomal Location of Human Ortholog: 6p21.3 Cellular Component: cell surface; cytoplasmic vesicle; cytosol; endoplasmic reticulum membrane; extracellular region; Golgi membrane; integral to plasma membrane; nucleoplasm; nucleus; plasma membrane Molecular Function:calcium ion binding; Notch binding; protein binding; protein heterodimerization activity; receptor activity Biological Process: activation of Notch receptor target transcription factor; cell differentiation; cell fate determination; embryonic development; endothelial cell morphogenesis; gene expression; hemopoiesis; mammary gland development; morphogenesis of a branching structure; negative regulation of cell differentiation; negative regulation of endothelial cell differentiation; negative regulation of Notch signaling pathway; Notch receptor processing; Notch signaling pathway; patterning of blood vessels; positive regulation of angiogenesis; positive regulation of transcription, DNA-dependent; regulation of protein localization; transcription initiation from RNA polymerase II promoter
NCBI Summary:	This gene encodes a member of the NOTCH family of proteins. Members of this Type I 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 signaling is an evolutionarily conserved intercellular signaling pathway that regulates interactions between physically adjacent cells through binding of Notch family receptors to their cognate ligands. The encoded preproprotein is proteolytically processed in the trans-Golgi network to generate two polypeptide chains that heterodimerize to form the mature cell-surface receptor. This receptor may play a role in vascular, renal and hepatic development. Mutations in this gene may be associated with schizophrenia. Alternative splicing results in multiple transcript variants, at least one of which encodes an isoform that is proteolytically processed. [provided by RefSeq, Jan 2016]
UniProt Code:	Q99466
NCBI GenInfo Identifier:	20139103
NCBI Gene ID:	4855
NCBI Accession:	Q99466.2
UniProt Secondary Accession:	Q99466,O00306, Q5SSY7, Q99458, Q99940, Q9H3S8, Q9UII9 Q9UIJ0, B0V183, B0V1X5,
UniProt Related Accession:	Q99466
Molecular Weight:	39,617 Da
NCBI Full Name:	Neurogenic locus notch homolog protein 4
NCBI Synonym Full Names:	notch 4
NCBI Official Symbol:	NOTCH4
NCBI Official Synonym Symbols:	INT3
NCBI Protein Information:	neurogenic locus notch homolog protein 4
UniProt Protein Name:	Neurogenic locus notch homolog protein 4
Protein Family:	Neurogenic locus notch homolog protein
UniProt Gene Name:	NOTCH4
UniProt Entry Name:	NOTC4_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 NOTCH4 ELISA Kit