Mouse Prostaglandin G/H synthase 2 (Ptgs2) ELISA Kit (MOEB0544)

Product Name:	Mouse Prostaglandin G/H synthase 2 (Ptgs2) ELISA Kit
SKU:	MOEB0544
Size:	96T
Target:	Mouse Prostaglandin G/H synthase 2 (Ptgs2)
Synonyms:	Cyclooxygenase-2, Glucocorticoid-regulated inflammatory cyclooxygenase, Gripghs, Macrophage activation-associated marker protein P71/73, PES-2, PHS II, Prostaglandin H2 synthase 2, Prostaglandin-endoperoxide synthase 2, TIS10 protein, COX-2, PGH synthase 2, Cox-2, Cox2, Pghs-b, Tis10
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.312-20ng/mL
Sensitivity:	0.165ng/mL

Intra CV:

5.1%

Inter CV:

9.3%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	89-89%	98-110%	87-99%	81-91%
EDTA Plasma(N=5)	99-110%	108-120%	97-107%	110-119%
Heparin Plasma(N=5)	88-99%	104-114%	107-116%	98-108%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	88	82-94
Plasma	90	84-96

Function:

Converts arachidonate to prostaglandin H2 (PGH2), a committed step in prostanoid synthesis. Constitutively expressed in some tissues in physiological conditions, such as the endothelium, kidney and brain, and in pathological conditions, such as in cancer. PTGS2 is responsible for production of inflammatory prostaglandins. Up-regulation of PTGS2 is also associated with increased cell adhesion, phenotypic changes, resistance to apoptosis and tumor angiogenesis. In cancer cells, PTGS2 is a key step in the production of prostaglandin E2 (PGE2), which plays important roles in modulating motility, proliferation and resistance to apoptosis.

Uniprot:	Q05769
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Prostaglandin G/H synthase 2
Sub Unit:	Homodimer.
Research Area:	Cardiovascular
Subcellular Location:	Microsome membrane Peripheral membrane protein Endoplasmic reticulum membrane Peripheral membrane protein
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	COX-2: Mediates the formation of prostaglandins from arachidonate. May have a role as a major mediator of inflammation and/or a role for prostanoid signaling in activity-dependent plasticity. Homodimer. Belongs to the prostaglandin G/H synthase family.
UniProt Protein Details:	Protein type:Lipid Metabolism - arachidonic acid; EC 1.14.99.1; Oxidoreductase Cellular Component: neuron projection; protein complex; membrane; intracellular membrane-bound organelle; endoplasmic reticulum; cytoplasm; caveola; nucleus Molecular Function:oxidoreductase activity, acting on single donors with incorporation of molecular oxygen, incorporation of two atoms of oxygen; prostaglandin-endoperoxide synthase activity; peroxidase activity; protein homodimerization activity; enzyme binding; dioxygenase activity; metal ion binding; heme binding; oxidoreductase activity; lipid binding Biological Process: positive regulation of nitric oxide biosynthetic process; positive regulation of apoptosis; positive regulation of smooth muscle cell proliferation; positive regulation of NF-kappaB import into nucleus; response to lipopolysaccharide; decidualization; sensory perception of pain; prostaglandin biosynthetic process; fatty acid metabolic process; positive regulation of synaptic transmission, glutamatergic; negative regulation of synaptic transmission, dopaminergic; bone mineralization; negative regulation of cell cycle; negative regulation of cell proliferation; regulation of blood pressure; negative regulation of smooth muscle contraction; positive regulation of cell proliferation; angiogenesis; positive regulation of smooth muscle contraction; inflammatory response; fatty acid biosynthetic process; negative regulation of calcium ion transport; cyclooxygenase pathway; positive regulation of synaptic plasticity; learning; memory; prostaglandin metabolic process; regulation of cell proliferation; ovulation; keratinocyte differentiation; response to cytokine stimulus; brown fat cell differentiation; positive regulation of vasoconstriction; positive regulation of fever; lipid metabolic process; response to oxidative stress; anagen; embryo implantation
UniProt Code:	Q05769
NCBI GenInfo Identifier:	118130137
NCBI Gene ID:	19225
NCBI Accession:	NP_035328.2
UniProt Secondary Accession:	Q05769,Q543K3,
UniProt Related Accession:	Q05769
Molecular Weight:	69,013 Da
NCBI Full Name:	Mus musculus prostaglandin-endoperoxide synthase 2 (Ptgs2), mRNA
NCBI Synonym Full Names:	prostaglandin-endoperoxide synthase 2
NCBI Official Symbol:	Ptgs2
NCBI Official Synonym Symbols:	COX2; Cox-2; PHS-2; Pghs2; TIS10; PGHS-2
NCBI Protein Information:	prostaglandin G/H synthase 2; PES-2; PGH synthase 2; PHS II; cyclooxygenase 2; cyclooxygenase-2; glucocorticoid-regulated inflammatory cyclooxygenase; gripghs; macrophage activation-associated marker protein P71/73; prostaglandin H2 synthase 2
UniProt Protein Name:	Prostaglandin G/H synthase 2
UniProt Synonym Protein Names:	Cyclooxygenase-2; COX-2; Glucocorticoid-regulated inflammatory cyclooxygenase; Gripghs; Macrophage activation-associated marker protein P71/73; PES-2; PHS II; Prostaglandin H2 synthase 2; PGH synthase 2; PGHS-2; Prostaglandin-endoperoxide synthase 2; TIS10 protein
UniProt Gene Name:	Ptgs2
UniProt Entry Name:	PGH2_MOUSE

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.

Mouse Prostaglandin G/H synthase 2 (Ptgs2) ELISA Kit (MOEB0544)

Description