The Mouse Leucine-rich Repeat Serine/Threonine-protein Kinase 2 (LRRK2) ELISA Kit is designed for the precise measurement of LRRK2 levels in mouse serum, plasma, and cell culture supernatants. This kit offers high sensitivity and specificity, ensuring accurate and consistent results for a variety of research applications.LRRK2 is an important protein involved in regulating cell signaling pathways and cell survival.
Dysregulation of LRRK2 has been linked to various diseases, including Parkinson's disease and other neurodegenerative disorders. As such, this ELISA kit is valuable for studying the role of LRRK2 in disease pathogenesis and for developing potential therapeutic interventions.
Positively regulates autophagy through a calcium-dependent activation of the CaMKK/AMPK signaling pathway. The process involves activation of nicotinic acid adenine dinucleotide phosphate (NAADP) receptors, increase in lysosomal pH, and calcium release from lysosomes. Together with RAB29, plays a role in the retrograde trafficking pathway for recycling proteins, such as mannose 6 phosphate receptor (M6PR), between lysosomes and the Golgi apparatus in a retromer-dependent manner. Regulates neuronal process morphology in the intact central nervous system (CNS). Phosphorylates PRDX3. Has GTPase activity.
Uniprot:
Q5S006
Sample Type:
Serum, plasma, tissue homogenates, cell culture supernates and other biological fluids
Specificity:
Natural and recombinant mouse Leucine-rich repeat serine/threonine-protein kinase 2
Sub Unit:
Homodimer. Interacts with PARK2, PRDX3 and TPCN2 (By similarity). Interacts with VPS35 and RAB29.
Research Area:
Neurosciences
Subcellular Location:
Membrane Peripheral membrane protein Cytoplasm Perikaryon Cell projection Axon Cell projection Dendrite Golgi apparatus Endoplasmic reticulum Cytoplasmic vesicle Secretory vesicle Synaptic vesicle membrane Peripheral membrane protein Cytoplasmic side Endosome Lysosome Mitochondrion outer membrane Mitochondrion inner membrane Mitochondrion matrix Localized in the cytoplasm and associated with cellular membrane structures. Colocalized with RAB29 along tubular structures emerging from Golgi apparatus. Localizes in intracytoplasmic punctate structures of neuronal perikarya and dendritic and axonal processes (By similarity). Predominantly associated with intracytoplasmic vesicular and membranous structures. Predominantly associated with the mitochondrial outer membrane of the mitochondria.
Storage:
Please see kit components below for exact storage details
Note:
For research use only
UniProt Protein Function:
LRRK2: a large multidomain protein kinase with a TKL-type kinase domain, multiple protein-protein interaction domains and a mitochondrial Rho domain (MIRO). May play a role in the etiology of Parkinson disease. May also have GTPase activity. Positively regulates autophagy through a calcium-dependent activation of the CaMKK/AMPK signaling pathway. The process involves activation of nicotinic acid adenine dinucleotide phosphate (NAADP) receptors, increase in lysosomal pH, and calcium release from lysosomes. Interacts with PARK2, PRDX3 and TPCN2. Expressed throughout the adult brain, but at a lower level than in heart and liver. Expressed in the cerebellum, cerebral cortex, medulla, spinal cord occipital pole, frontal lobe, temporal lobe and putamen. Expression is particularly high in brain dopaminoceptive areas. Defects in LRRK2 are the cause of Parkinson disease type 8 (PARK8). A slowly progressive neurodegenerative disorder characterized by bradykinesia, rigidity, resting tremor, postural instability, neuronal loss in the substantia nigra, and the presence of neurofibrillary MAPT (tau)-positive and Lewy bodies in some patients.Protein type: Protein kinase, Ser/Thr (non-receptor); Protein kinase, TKL; EC 2.7.11.1; Kinase, protein; TKL group; LRRK familyChromosomal Location of Human Ortholog: 12q12Cellular Component: dendrite cytoplasm; Golgi apparatus; extracellular space; neuron projection; synaptic vesicle membrane; mitochondrion; lysosome; endoplasmic reticulum; dendrite; terminal button; perikaryon; trans-Golgi network; inclusion body; cytosol; lipid raft; mitochondrial outer membrane; cell soma; axon; mitochondrial matrix; cytoplasm; mitochondrial membrane; mitochondrial inner membrane; plasma membrane; cytoplasmic vesicle; cell junction; endosomeMolecular Function: GTPase activity; tubulin binding; identical protein binding; clathrin binding; GTP-dependent protein kinase activity; protein homodimerization activity; Rho GTPase binding; GTP binding; actin binding; GTPase activator activity; protein kinase activity; MAP kinase kinase activity; protein serine/threonine kinase activity; protein binding; SNARE binding; protein kinase A binding; syntaxin-1 binding; kinase activity; glycoprotein binding; ATP bindingBiological Process: regulation of synaptic transmission, glutamatergic; activation of MAPKK activity; positive regulation of protein binding; activation of MAPK activity; protein amino acid autophosphorylation; regulation of neuron maturation; determination of adult life span; protein amino acid phosphorylation; positive regulation of MAP kinase activity; tangential migration from the subventricular zone to the olfactory bulb; negative regulation of protein amino acid phosphorylation; regulation of dopamine receptor signaling pathway; small GTPase mediated signal transduction; regulation of mitochondrial depolarization; negative regulation of protein binding; positive regulation of autophagy; neuromuscular junction development; positive regulation of dopamine receptor signaling pathway; olfactory bulb development; peptidyl-threonine phosphorylation; MAPKKK cascade; endocytosis; regulation of locomotion; positive regulation of programmed cell death; regulation of membrane potential; peptidyl-serine phosphorylation; positive regulation of protein ubiquitination; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; intracellular distribution of mitochondria; autophagy; positive regulation of protein amino acid phosphorylation; response to oxidative stress; regulation of excitatory postsynaptic membrane potential; neurite morphogenesis; positive regulation of GTPase activityDisease: Parkinson Disease 8, Autosomal Dominant
UniProt Protein Details:
NCBI Summary:
This gene is a member of the leucine-rich repeat kinase family and encodes a protein with an ankryin repeat region, a leucine-rich repeat (LRR) domain, a kinase domain, a DFG-like motif, a RAS domain, a GTPase domain, a MLK-like domain, and a WD40 domain. The protein is present largely in the cytoplasm but also associates with the mitochondrial outer membrane. Mutations in this gene have been associated with Parkinson disease-8. [provided by RefSeq, Jul 2008]
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.
