The Mouse 85 kDa Calcium-Independent Phospholipase A2 (PLA2G6) ELISA Kit is a powerful tool for researchers looking to accurately measure levels of PLA2G6 in mouse serum, plasma, and cell culture supernatants. This kit offers exceptional sensitivity and specificity, ensuring the precision and consistency of your results, making it perfect for a variety of investigative purposes.PLA2G6 is a key enzyme involved in lipid metabolism and cell signaling, playing a crucial role in various biological processes.
Dysregulation of PLA2G6 has been linked to neurological disorders, neuroinflammation, and neurodegeneration, making it an important target for research and drug development.With the Mouse 85 kDa PLA2G6 ELISA Kit, researchers can easily and reliably measure PLA2G6 levels in their samples, paving the way for a better understanding of its role in health and disease.
Group VI phospholipase A2, Intracellular membrane-associated calcium-independent phospholipase A2 beta, Patatin-like phospholipase domain-containing protein 9, GVI PLA2, iPLA2-beta, PNPLA9, CaI-PLA2, Pnpla9
Assay Type:
Sandwich
Detection Method:
ELISA
Reactivity:
Mouse
Detection Range:
0.312-20ng/mL
Sensitivity:
0.183ng/mL
Intra CV:
Provided with the Kit
Inter CV:
Provided with the Kit
Linearity:
Provided with the Kit
Recovery:
Provided with the Kit
Function:
Catalyzes the release of fatty acids from phospholipids. It has been implicated in normal phospholipid remodeling, nitric oxide-induced or vasopressin-induced arachidonic acid release and in leukotriene and prostaglandin production. May participate in fas mediated apoptosis and in regulating transmembrane ion flux in glucose-stimulated B-cells. Has a role in cardiolipin (CL) deacylation. Required for both speed and directionality of monocyte MCP1/CCL2-induced chemotaxis through regulation of F-actin polymerization at the pseudopods.
Uniprot:
P97819
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse 85/88 kDa calcium-independent phospholipase A2
Subcellular Location:
Isoform Short Cytoplasm
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
PLA2G6: Catalyzes the release of fatty acids from phospholipids. It has been implicated in normal phospholipid remodeling, nitric oxide-induced or vasopressin-induced arachidonic acid release and in leukotriene and prostaglandin production. May participate in fas mediated apoptosis and in regulating transmembrane ion flux in glucose-stimulated B-cells. Has a role in cardiolipin (CL) deacylation. Required for both speed and directionality of monocyte MCP1/CCL2-induced chemotaxis through regulation of F- actin polymerization at the pseudopods. Defects in PLA2G6 are the cause of neurodegeneration with brain iron accumulation type 2B (NBIA2B). A neurodegenerative disorder associated with iron accumulation in the brain, primarily in the basal ganglia. It is characterized by progressive extrapyramidal dysfunction leading to rigidity, dystonia, dysarthria and sensorimotor impairment. Defects in PLA2G6 are the cause of neurodegeneration with brain iron accumulation type 2A (NBIA2A); also known as Seitelberger disease. NBIA2A is a neurodegenerative disease characterized by pathologic axonal swelling and spheroid bodies in the central nervous system. Onset is within the first 2 years of life with death by age 10 years. Defects in PLA2G6 are the cause of Parkinson disease type 14 (PARK14). An adult-onset progressive neurodegenerative disorder characterized by parkinsonism, dystonia, severe cognitive decline, cerebral and cerebellar atrophy and absent iron in the basal ganglia on magnetic resonance imaging. 4 isoforms of the human protein are produced by alternative splicing.Protein type: Lipid Metabolism - ether lipid; Lipid Metabolism - linoleic acid; Lipid Metabolism - alpha-linolenic acid; Lipid Metabolism - glycerophospholipid; EC 3.1.1.4; Phospholipase; Lipid Metabolism - arachidonic acidCellular Component: mitochondrion; membrane; cytoplasm; microtubule organizing center; cytosolMolecular Function: calmodulin binding; phospholipase A2 activity; ATP-dependent protein binding; calcium-independent phospholipase A2 activity; hydrolase activity; protein kinase bindingBiological Process: cardiolipin biosynthetic process; urinary bladder smooth muscle contraction; elevation of cytosolic calcium ion concentration; metabolic process; negative regulation of synaptic transmission, glutamatergic; lipid metabolic process; positive regulation of protein amino acid phosphorylation; chemotaxis; positive regulation of vasodilation; memory; lipid catabolic process; positive regulation of exocytosis
85/88 kDa calcium-independent phospholipase A2; GVI PLA2; caI-PLA2; iPLA2-beta; group VI phospholipase A2; 85 kDa calcium-independent phospholipase A2; patatin-like phospholipase domain-containing protein 9; intracellular membrane-associated calcium-independent phospholipase A2 beta
UniProt Protein Name:
85/88 kDa calcium-independent phospholipase A2
UniProt Synonym Protein Names:
Group VI phospholipase A2; GVI PLA2; Intracellular membrane-associated calcium-independent phospholipase A2 beta; iPLA2-beta; Patatin-like phospholipase domain-containing protein 9
Protein Family:
UniProt Gene Name:
Pla2g6
UniProt Entry Name:
PLPL9_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.
