Rat Nos3 ELISA Kit (RTEB0466)- High Sensitivity

Product Name:	Rat Nitric oxide synthase, endothelial (Nos3) ELISA Kit
SKU:	RTEB0466
Size:	96T
Target:	Rat Nitric oxide synthase, endothelial (Nos3)
Synonyms:	Constitutive NOS, EC-NOS, Endothelial NOS, NOS type III, cNOS, eNOS, NOSIII
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Rat
Detection Range:	0.156-10ng/ml
Sensitivity:	0.078ng/mL

Intra CV:

4.6%

Inter CV:

8.6%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	80-90%	84-93%	100-110%	93-102%
EDTA Plasma(N=5)	100-110%	96-106%	98-107%	95-104%
Heparin Plasma(N=5)	96-104%	104-114%	107-119%	95-106%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	103	97-109
Plasma	105	99-111

Function:

Produces nitric oxide (NO) which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets.

Uniprot:	Q62600
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant rat Nitric oxide synthase, endothelial
Sub Unit:	Homodimer. Interacts with NOSIP and NOSTRIN (By similarity). Interacts with HSP90AB1.
Research Area:	Cancer
Subcellular Location:	Membrane Caveola Cytoplasm Cytoskeleton Golgi apparatus Cell membrane Specifically associates with actin cytoskeleton in the G2 phase of the cell cycle, which is favored by interaction with NOSIP and results in a reduced enzymatic activity.
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	eNOS: endothelial constitutive nitric oxide synthase. Synthesizes nitric oxide (NO) from arginine and oxygen, which is implicated in vascular smooth muscle relaxation through a cGMP-mediated signal transduction pathway. NO mediates vascular endothelial growth factor (VEGF)-induced angiogenesis in coronary vessels and promotes blood clotting through the activation of platelets. Induced in humans by caloric restriction. Stimulated by calcium/calmodulin. Inhibited by NOSIP and NOSTRIN.
UniProt Protein Details:	Protein type:EC 1.14.13.39; Oxidoreductase; Amino Acid Metabolism - arginine and proline Cellular Component: apical part of cell; caveola; cytoplasm; cytoskeleton; cytosol; Golgi apparatus; lipid raft; mitochondrion; nucleolus; nucleus; plasma membrane; sarcolemma; vesicle membrane Molecular Function:actin binding; actin monomer binding; beta-catenin binding; cadherin binding; calmodulin binding; FAD binding; FMN binding; heme binding; Hsp90 protein binding; iron ion binding; NADP binding; nitric-oxide synthase activity; nitric-oxide synthase binding; protein binding Biological Process: aging; angiogenesis; arginine catabolic process; blood vessel remodeling; endothelial cell migration; female pregnancy; in utero embryonic development; lipopolysaccharide-mediated signaling pathway; lung development; negative regulation of blood pressure; negative regulation of calcium ion transport; negative regulation of cell proliferation; negative regulation of hydrolase activity; negative regulation of muscle hyperplasia; negative regulation of potassium ion transport; negative regulation of smooth muscle cell proliferation; nitric oxide biosynthetic process; nitric oxide mediated signal transduction; ovulation from ovarian follicle; positive regulation of angiogenesis; positive regulation of apoptosis; positive regulation of guanylate cyclase activity; positive regulation of vasodilation; regulation of blood vessel size; regulation of sodium ion transport; regulation of systemic arterial blood pressure by endothelin; regulation of the force of heart contraction by chemical signal; removal of superoxide radicals; response to activity; response to amino acid stimulus; response to axon injury; response to corticosterone stimulus; response to cytokine stimulus; response to drug; response to estradiol stimulus; response to ethanol; response to fructose stimulus; response to hyperoxia; response to hypoxia; response to lead ion; response to lipopolysaccharide; response to lipoprotein stimulus; response to mechanical stimulus; response to metal ion; response to organic cyclic substance; response to peptide hormone stimulus; retina development in camera-type eye; signal transduction; smooth muscle hyperplasia
NCBI Summary:	enzyme that synthesizes Nitric oxide from L-arginine [RGD, Feb 2006]
UniProt Code:	Q62600
NCBI GenInfo Identifier:	46402488
NCBI Gene ID:	24600
NCBI Accession:	NP_068610.1
UniProt Secondary Accession:	Q62600,O88672, O89041, Q62734, Q68GV6, Q75NE4,
UniProt Related Accession:	Q62600
Molecular Weight:	133,290 Da
NCBI Full Name:	nitric oxide synthase, endothelial
NCBI Synonym Full Names:	nitric oxide synthase 3
NCBI Official Symbol:	Nos3
NCBI Official Synonym Symbols:	eNos
NCBI Protein Information:	nitric oxide synthase, endothelial
UniProt Protein Name:	Nitric oxide synthase, endothelial
UniProt Synonym Protein Names:	Constitutive NOS; cNOS; EC-NOS; Endothelial NOS; eNOS; NOS type III; NOSIII
Protein Family:	Nitric oxide synthase
UniProt Gene Name:	Nos3
UniProt Entry Name:	NOS3_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.

ELISA	Antibodies
Rat eNOS / Nitric oxide synthase, endothelial ELISA Kit	SOX2 Antibody, Biotin conjugated (PACO33707)
Rat NOS3/eNOS (Nitric Oxide Synthase 3, Endothelial) CLIA Kit (RTES00200)	Nos3 Antibody (PACO33708)
	Nos3 Antibody, HRP conjugated (PACO33709)
	Nos3 Antibody, FITC conjugated (PACO33710)