Mouse Pla2g2a ELISA Kit (MOEB2337)- High Sensitivity

Product Name:	Mouse Phospholipase A2, membrane associated (Pla2g2a) ELISA Kit
SKU:	MOEB2337
Size:	96T
Target:	Mouse Phospholipase A2, membrane associated (Pla2g2a)
Synonyms:	Enhancing factor, GIIC sPLA2, Group IIA phospholipase A2, Phosphatidylcholine 2-acylhydrolase 2A, EF
Assay Type:	Sandwich
Detection Method:	ELISA
Reactivity:	Mouse
Detection Range:	0.625-40ng/mL
Sensitivity:	0.319ng/mL

Intra CV:

7.4%

Inter CV:

9.0%

Linearity:

Sample	1:2	1:4	1:8	1:16
Serum(N=5)	103-113%	94-104%	104-113%	103-113%
EDTA Plasma(N=5)	101-111%	92-102%	103-112%	97-107%
Heparin Plasma(N=5)	98-108%	100-110%	92-102%	97-107%

Recovery:

Sample Type	Average(%)	Recovery Range(%)
Serum	100	94-106
Plasma	102	96-108

Function:

Catalyzes the calcium-dependent hydrolysis of the 2-acyl groups in 3-sn-phosphoglycerides (PubMed:8425615). Thought to participate in the regulation of phospholipid metabolism in biomembranes including eicosanoid biosynthesis. Independent of its catalytic activity, acts as a ligand for integrins. Binds to and activates integrins ITGAV:ITGB3, ITGA4:ITGB1 and ITGA5:ITGB. Binds to a site (site 2) which is distinct from the classical ligand-binding site (site 1) and induces integrin conformational changes and enhanced ligand binding to site 1. Induces cell proliferation in an integrin-dependent manner.

Uniprot:	P31482
Sample Type:	Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:	Natural and recombinant mouse Phospholipase A2, membrane associated
Research Area:	Cardiovascular
Subcellular Location:	Cell membrane Peripheral membrane protein Secreted
Storage:	Please see kit components below for exact storage details
Note:	For research use only

UniProt Protein Function:	PLA2G2A: Thought to participate in the regulation of the phospholipid metabolism in biomembranes including eicosanoid biosynthesis. Catalyzes the calcium-dependent hydrolysis of the 2- acyl groups in 3-sn-phosphoglycerides. Belongs to the phospholipase A2 family.Protein type: Secreted, signal peptide; Lipid Metabolism - ether lipid; Phospholipase; Secreted; Mitochondrial; Lipid Metabolism - alpha-linolenic acid; Lipid Metabolism - arachidonic acid; Vesicle; Lipid Metabolism - linoleic acid; Lipid Metabolism - glycerophospholipid; EC 3.1.1.4; Endoplasmic reticulumCellular Component: membrane; mitochondrionMolecular Function: phospholipase A2 activity; hydrolase activity; metal ion binding; calcium ion bindingBiological Process: negative regulation of cell proliferation; somatic stem cell maintenance; phospholipid metabolic process; regulation of growth; lipid metabolic process; regulation of epithelial cell proliferation; lipid catabolic process; regulation of cell proliferation; negative regulation of epithelial cell proliferation
UniProt Protein Details:
NCBI Summary:	Proteins belonging to the phospholipase A2 (PLA2) family hydrolyze phospholipids into sn2 fatty acids and lysophospholipids. They function in a variety of cellular processes, including the digestion of phospholipids and the production of molecules that induce inflammatory responses. This gene encodes a member of the group II class of secretory PLA2s. The secreted enzyme binds to heparin on the cell surface. Mutations in this gene increase the occurrence of intestinal polyps caused by a dominant mutation in the adenomatosis polyposis coli gene. A frameshift inactivates this gene product in some mouse strains including the strain of the reference genome, C57BL/6J, whereas a functional protein is produced in other strains. [provided by RefSeq, Jul 2008]
UniProt Code:	P31482
NCBI GenInfo Identifier:	132626633
NCBI Gene ID:	18780
NCBI Accession:	NP_001076000.1
UniProt Secondary Accession:	P31482,Q60871
UniProt Related Accession:	P31482
Molecular Weight:	16,145 Da
NCBI Full Name:	phospholipase A2, membrane associated
NCBI Synonym Full Names:	phospholipase A2, group IIA (platelets, synovial fluid)
NCBI Official Symbol:	Pla2g2a
NCBI Official Synonym Symbols:	EF; Mom1; Pla2; sPLA2; sPla2-IIA
NCBI Protein Information:	phospholipase A2, membrane associated
UniProt Protein Name:	Phospholipase A2, membrane associated
UniProt Synonym Protein Names:	Enhancing factor; EF; GIIC sPLA2; Group IIA phospholipase A2; Phosphatidylcholine 2-acylhydrolase 2A
Protein Family:	Phospholipase
UniProt Gene Name:	Pla2g2a
UniProt Entry Name:	PA2GA_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.