eNOS (Phospho-Ser1176) Fluorometric Cell-Based ELISA Kit

SKU:
FBCAB00064
Product type:
ELISA Kit
ELISA Type:
Cell Based
€599

Description

Technical Manual

eNOS (Phospho-Ser1176) Cell-Based ELISA Kit

The eNOS (Phospho-Ser1176) Cell-Based ELISA Kit is a convenient, lysate- free, high throughput and sensitive assay kit that can monitor eNOS phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated eNOS in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals), or activators have on eNOS phosphorylation.

How does our eNOS (Phospho-Ser1176) Fluorometric Cell-Based ELISA Kit work?

Qualitative determination of eNOS (Phospho-Ser1176) concentration is achieved by an indirect ELISA format. In essence, eNOS (Phospho-Ser1176) is captured by eNOS (Phospho-Ser1176)-specific primary (1°) antibodies while Dye 1-conjugated and Dye 2-conjugated secondary (2°) antibodies bind the Fc region of the 1° antibody. Through this binding, the dye conjugated to the 2° antibody can emit light at a certain wavelength given proper excitation, hence allowing for a fluorometric detection method. Due to the qualitative nature of the Cell-Based ELISA, multiple normalization methods are needed:

1. A monoclonal antibody specific for human GAPDH is included to serve as an internal positive control in normalizing the target RFU values.
2. An antibody against the nonphosphorylated counterpart of eNOS (Phospho-Ser1176) is also provided for normalization purposes. The RFU values obtained for non-phosphorylated eNOS can be used to normalize the RFU value for phosphorylated eNOS.

eNOS (Phospho-Ser1176) Fluorometric Cell-Based ELISA Kit -Information

Product Name:eNOS (Phospho-Ser1176) Fluorometric Cell-Based ELISA Kit
Product Code/SKU:FBCAB00064
Description:The eNOS (Phospho-Ser1176) Fluorometric Cell-Based Phospho ELISA Kit is a convenient, lysate-free, high throughput and sensitive assay kit that can monitor eNOS (Phospho-Ser1176) protein phosphorylation and expression profile in cells. The kit can be used for measuring the relative amounts of phosphorylated eNOS (Phospho-Ser1176) in cultured cells as well as screening for the effects that various treatments, inhibitors (ie. siRNA or chemicals, or activators have on NOS3 phosphorylation.
Dynamic Range:> 5000 Cells
Detection Method:Fluorometric
Storage/Stability:4°C/6 Months
Reactivity:Human, Mouse, Rat
Assay Type:Cell-Based ELISA
Database Links:Gene ID: 4846, UniProt ID: P29474, OMIM #: 163729, Unigene #: Hs.647092
Format:Two 96-Well Plates
NCBI Gene Symbol:NOS3
Sub Type:Phospho
Target Name:Phospho-eNOS (Ser1176)

Kit Principle

Figure: Schematic representation of Assay Genie Cell-Based Fluorometric ELISA principle

Kit components

Quantity

96-Well Black Cell CultureClear-Bottom Microplate2 plates
10X TBS24 ml
Quenching Buffer24 ml
Blocking Buffer50 ml
15X Wash Buffer50 ml
Primary Antibody Diluent12 ml
100x Anti-Phospho Target Antibody 60 µl
100x Anti-Target Antibody60 µl
Anti-GAPDH Antibody110 µl
Dye-1 Conjugated Anti-Rabbit IgG Antibody6 ml
Dye-2 Conjugated Anti-Mouse IgG Antibody6 ml
Adhesive Plate Seals2 seals

Additional equipment and materials required

The following materials and/or equipment are NOT provided in this kit but are necessary to successfully conduct the experiment:

  • Fluorescent plate reader with two channels at Ex/Em: 651/667 and 495/521
  • Micropipettes capable of measuring volumes from 1 µl to 1 ml
  • Deionized or sterile water (ddH2O)
  • 37% formaldehyde (Sigma Cat# F-8775) or formaldehyde from other sources
  • Squirt bottle, manifold dispenser, multichannel pipette reservoir or automated microplate washer
  • Graph paper or computer software capable of generating or displaying logarithmic functions
  • Absorbent papers or vacuum aspirator
  • Test tubes or microfuge tubes capable of storing ≥1 ml
  • Poly-L-Lysine (Sigma Cat# P4832 for suspension cells)
  • Orbital shaker (optional)

Kit Protocol

This is a summarized version of the kit protocol. Please view the technical manual of this kit for information on sample preparation, reagent preparation and plate lay out.

1.Seed 200 µl of desired cell concentration in culture medium into each well of the 96-well plates. For suspension cells and loosely attached cells, coat the plates with 100 µl of 10 µg/ml Poly-L-Lysine (not included) to each well of a 96-well plate for 30 minutes at 37°C prior to adding cells.
2.Incubate the cells for overnight at 37°C, 5% CO2.
3.Treat the cells as desired.
4.Remove the cell culture medium and rinse with 200 µl of 1x TBS, twice.
5.Fix the cells by incubating with 100 µl of Fixing Solution for 20 minutes at room temperature. The 4% formaldehyde is used for adherent cells and 8% formaldehyde is used for suspension cells and loosely attached cells.
6.Remove the Fixing Solution and wash the plate 3 times with 200 µl 1x Wash Buffer for 3 minutes. The plate can be stored at 4°C for a week.
7.Add 100 µl of Quenching Buffer and incubate for 20 minutes at room temperature.
8.Wash the plate 3 times with 1x Wash Buffer for 3 minutes each time.
9.Dispense 200 µl of Blocking Buffer and incubate for 1 hour at room temperature.
10. Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
11.Add 50 µl of Primary Antibody Mixture P to corresponding wells for eNOS (Phospho-Ser1176) detection. Add 50 µl of Primary Antibody Mixture NP to the corresponding wells for total eNOS detection. Cover the plate with parafilm and incubate for 16 hours (overnight) at 4°C. If the target expression is known to be high, incubate for 2 hours at room temperature.
12.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
13.Add 50 ul of Secondary Antibody Mixture to corresponding wells and incubate for 1.5 hours at room temperature in the dark.
14.Wash 3 times with 200 µl of 1x Wash Buffer for 3 minutes each time.
15.Read the plate(s) at Ex/Em: 651/667 (Dye 1) and 495/521 (Dye 2). Shield plates from direct light exposure.
16.Wash 3 times with 200 µl of 1x Wash Buffer for 5 minutes each time.

eNOS (Phospho-Ser1176) - Protein Information

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:Amino Acid Metabolism - arginine and proline; Oxidoreductase; EC 1.14.13.39

Chromosomal Location of Human Ortholog: 7q36

Cellular Component: caveola; cytoplasm; cytoskeleton; cytosol; Golgi membrane; nucleus; plasma membrane

Molecular Function:actin monomer binding; cadmium ion binding; calmodulin binding; FAD binding; FMN binding; heme binding; iron ion binding; NADP binding; nitric-oxide synthase activity; protein binding

Biological Process: angiogenesis; arginine catabolic process; blood coagulation; blood vessel remodeling; cell redox homeostasis; endothelial cell migration; in utero embryonic development; lipopolysaccharide-mediated signaling pathway; lung development; mitochondrion organization and biogenesis; 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; nitric oxide biosynthetic process; nitric oxide mediated signal transduction; nitric oxide metabolic process; ovulation from ovarian follicle; positive regulation of angiogenesis; positive regulation of guanylate cyclase activity; positive regulation of vasodilation; regulation of blood pressure; regulation of blood vessel size; regulation of nitric-oxide synthase activity; 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 heat; smooth muscle hyperplasia; vascular endothelial growth factor receptor signaling pathway

Disease: Alzheimer Disease; Hypertension, Essential; Preeclampsia/eclampsia 1; Stroke, Ischemic

NCBI Summary:Nitric oxide is a reactive free radical which acts as a biologic mediator in several processes, including neurotransmission and antimicrobial and antitumoral activities. Nitric oxide is synthesized from L-arginine by nitric oxide synthases. Variations in this gene are associated with susceptibility to coronary spasm. Multiple transcript variants encoding different isoforms have been found for this gene. [provided by RefSeq, May 2009]
UniProt Code:P29474
NCBI GenInfo Identifier:266648
NCBI Gene ID:4846
NCBI Accession:P29474.3
UniProt Secondary Accession:P29474,Q13662, Q14251, Q14434, Q548C1, Q6GSL5, Q9UDC6 A0S0A7, A0S0A8, A8KA63, B2RCQ1, E9PFR2,
UniProt Related Accession:P29474
Molecular Weight:
NCBI Full Name:Nitric oxide synthase, endothelial
NCBI Synonym Full Names:nitric oxide synthase 3
NCBI Official Symbol:NOS3  
NCBI Official Synonym Symbols:eNOS; ECNOS  
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_HUMAN