Product Name:	Human Endothelin-1 ELISA Kit
Product Code:	HUFI00466
Size:	96 Assays
Alias:	ET-1, EDN1, ET1, HDLCQ7, PPET1, Preproendothelin-1, endothelin 1, endothelin-1, preproendothelin-1
Detection method:	Sandwich ELISA, Double Antibody
Application:	This immunoassay kit allows for the in vitro quantitative determination of Human EDN1 concentrations in serum plasma and other biological fluids.
Sensitivity:	0.75pg/ml
Range:	1.25-80pg/ml
Storage:	4°C for 6 months
Note:	For Research Use Only

Recovery:

Matrices listed below were spiked with certain level of Human EDN1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human EDN1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-105	98
EDTA plasma(n=5)	87-100	94
UFH plasma(n=5)	85-102	95

Linearity:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human EDN1 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)	85-103%	87-101%	90-104%
EDTA plasma(n=5)	82-92%	83-101%	87-100%
UFH plasma(n=5)	87-99%	81-99%	81-98%

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

P05305

UniProt Protein Function:	EDN1: Endothelins are endothelium-derived vasoconstrictor peptides. Belongs to the endothelin/sarafotoxin family.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Secreted Chromosomal Location of Human Ortholog: 6p24.1 Cellular Component: extracellular space; cytoplasm; extracellular region Molecular Function:protein binding; hormone activity; endothelin B receptor binding; cytokine activity; endothelin A receptor binding Biological Process: response to nicotine; positive regulation of JNK activity; elevation of cytosolic calcium ion concentration during G-protein signaling, coupled to IP3 second messenger (phospholipase C activating); positive regulation of nitric oxide biosynthetic process; regulation of systemic arterial blood pressure by endothelin; heart development; response to lipopolysaccharide; middle ear morphogenesis; prostaglandin biosynthetic process; sensory perception of pain; positive regulation of MAP kinase activity; negative regulation of cAMP biosynthetic process; elevation of cytosolic calcium ion concentration; cell surface receptor linked signal transduction; cell-cell signaling; negative regulation of nitric-oxide synthase biosynthetic process; protein kinase C activation; cell growth; neutrophil chemotaxis; rhythmic excitation; positive regulation of mitosis; negative regulation of blood coagulation; positive regulation of heart rate; respiratory gaseous exchange; response to testosterone stimulus; response to amino acid stimulus; peptide hormone secretion; leukocyte activation; patterning of blood vessels; membrane depolarization; protein kinase C deactivation; regulation of vasoconstriction; positive regulation of transcription from RNA polymerase II promoter; response to activity; superoxide release; positive regulation of odontogenesis; epithelial fluid transport; phosphoinositide 3-kinase cascade; G-protein signaling, phospholipase D activating pathway; neural crest cell development; positive regulation of cell size; negative regulation of hormone secretion; positive regulation of smooth muscle cell proliferation; negative regulation of cellular protein metabolic process; glucose transport; negative regulation of transcription from RNA polymerase II promoter; vein smooth muscle contraction; histamine secretion; nitric oxide transport; positive regulation of cell proliferation; regulation of pH; positive regulation of smooth muscle contraction; artery smooth muscle contraction; vasoconstriction; inositol phosphate-mediated signaling; in utero embryonic development; calcium-mediated signaling; positive regulation of hormone secretion; multicellular organismal aging; body fluid secretion; G-protein coupled receptor protein signaling pathway; dorsal/ventral pattern formation; cartilage development; response to ozone; positive regulation of prostaglandin secretion; maternal process involved in parturition; regulation of sensory perception of pain; positive regulation of cell migration Disease: Question Mark Ears, Isolated; Auriculocondylar Syndrome 3
NCBI Summary:	The protein encoded by this gene is proteolytically processed to release a secreted peptide termed endothelin 1. This peptide is a potent vasoconstrictor and is produced by vascular endothelial cells. Endothelin 1 also can affect the central nervous system. Two transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, Nov 2009]
UniProt Code:	P05305
NCBI GenInfo Identifier:	119610
NCBI Gene ID:	1906
NCBI Accession:	P05305.1
UniProt Secondary Accession:	P05305,Q96DA1,
UniProt Related Accession:	P05305
Molecular Weight:	24,425 Da
NCBI Full Name:	Endothelin-1
NCBI Synonym Full Names:	endothelin 1
NCBI Official Symbol:	EDN1
NCBI Official Synonym Symbols:	ET1; QME; PPET1; ARCND3; HDLCQ7
NCBI Protein Information:	endothelin-1; preproendothelin-1
UniProt Protein Name:	Endothelin-1
UniProt Synonym Protein Names:	Preproendothelin-1; PPET1
Protein Family:	Endothelin
UniProt Gene Name:	EDN1
UniProt Entry Name:	EDN1_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 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.

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Human Endothelin-1 ELISA Kit

Description