Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit

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SKU:
HUFI01480
€599

Description

ELISA Kit Technical ManualELISA Kit Technical ManualMSDS

Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit - Information

The ELISA Genie LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit can assay for LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 in the following samples: serum, blood, plasma, cell culture supernatant and other related supernatants and tissues.

How our LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kits Work?

The ELISA Genie (enzyme-linked immunosorbent assays) assay kits are designed for the quantitative measurement of analytes in a wide variety of samples. As today's scientists demand high quality consistent data for high impact journals, ELISA Genie have developed our range of sensitive, fast and reliable ELISA kit assays to meet and exceed those demands. Our assay kits use a quantitative sandwich ELISA technique and each kit comes with highly specific antibodies pre-coated onto a 96-well microtiter plate.

At ELISA Genie we understand the need for speed! Therefore, we have developed an ultra-fast protocol meaning you achieve your results rapidly. So, once you have prepared and plated your samples, blanks and standards, you simply incubate with a highly specific biotin-conjugated primary antibody and Avidin conjugated to Horseradish Peroxidase (HRP) and incubate for the appropriate length of time. After washing the plate according to the protocol and addition of the TMB (3,3',5,5'-Tetramethylbenzidine) solution, the appearance of a blue colour should be detected due to an enzymatic reaction catalysed by HRP. Next step is the addition of the Stop Solution which terminates the HRP reaction and the blue colour turns yellow with the signal intensity measured on a plate reader at 450nm. The amount of bound LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 is proportional to the signal generated by the reaction meaning the kit assay gives you a quantitative measurement of the analyte in your samples.

Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit - Data

Description

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). Plays a role in synaptic vesicle trafficking. Phosphorylates PRDX3. Has GTPase activity. May play a role in the phosphorylation of proteins central to Parkinson disease.

Post-Translational Modification

Autophosphorylated.

Uniprot ID Q5S007
Alias

LRRK2/AURA17/Dardarin/PARK8/ROCO2/augmented in rheumatoid arthritis 17/leucine-rich repeat kinase 2/leucine-rich repeat serinethreonine-protein kinase 2/RIPK7

Detection method

Sandwich ELISA Double Antibody

Application

This immunoassay kit allows for the in vitro quantitative determination of LRRK2 concentrations in serum plasma and other biological fluids.

Size

96T

Range

0.156-10ng/ml

Sensitivity

< 0.094ng/ml

Storage

4'C for 6 months

Recovery

Matrices listed below were spiked with certain level of LRRK2 and the recovery rates were calculated by comparing the measured value to the expected amount of LRRK2 in samples.

Matrix Recovery range(%) Average(%)
serum(n=5) 86-100 94
EDTA plasma(n=5) 86-105 96
UFH plasma(n=5) 87-104 96
Linearity

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of LRRK2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample 1:2 1:4 1:8 1:16
serum(n=5) 87-97% 87-104% 86-93% 86-100%
EDTA plasma(n=5) 83-97% 82-92% 84-95% 87-101%
UFH plasma(n=5) 83-100% 83-100% 81-93% 80-96%
CV(%)

Intra-Assay: CV<8%
Inter-Assay: CV<10%

Note

For Research Use Only

Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit Protocol

The below protocol is a sample protocol for Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit using a biotinylated detection antibody and streptavidin-HRP. Sandwich ELISAs allow for the detection and quantification of an analyte in a sample by using known analyte concentrations as standards and plotting absorbance of known concentrations vs known standard concentrations. This allows the researcher to calculate the amount of Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 present in their sample.

Before adding to wells, equilibrate the SABC working solution and TMB substrate for at least 30 min at 37 °C. When diluting samples and reagents, they must be mixed completely and evenly. It is recommended to plot a standard curve for each test.

Sandwich Protocol

Sandwich ELISA Protocol

Kit Protocol:

1.Set standard, test sample and control (zero) wells on the pre-coated plate respectively, and then, record their positions. It is recommended to measure each standard and sample in duplicate. Wash plate 2 times before adding standard, sample and control (zero) wells!
2.Aliquot 0.1ml standard solutions into the standard wells.
3.Add 0.1 ml of Sample / Standard dilution buffer into the control (zero) well.
4.Add 0.1 ml of properly diluted sample ( Human serum, plasma, tissue homogenates and other biological fluids.) into test sample wells.
5.Seal the plate with a cover and incubate at 37 °C for 90 min.
6.Remove the cover and discard the plate content, clap the plate on the absorbent filter papers or other absorbent material. Do NOT let the wells completely dry at any time. Wash plate X2.
7.Add 0.1 ml of Biotin- detection antibody working solution into the above wells (standard, test sample & zero wells). Add the solution at the bottom of each well without touching the side wall.
8.Seal the plate with a cover and incubate at 37°C for 60 min.
9.Remove the cover, and wash plate 3 times with Wash buffer. Let wash buffer rest in wells for 1 min between each wash.
10.Add 0.1 ml of SABC working solution into each well, cover the plate and incubate at 37°C for 30 min.
11.Remove the cover and wash plate 5 times with Wash buffer, and each time let the wash buffer stay in the wells for 1-2 min.
12.Add 90 µl of TMB substrate into each well, cover the plate and incubate at 37°C in dark within 10-20 min. (Note: This incubation time is for reference use only, the optimal time should be determined by end user.) And the shades of blue can be seen in the first 3-4 wells (with most concentrated standard solutions), the other wells show no obvious color.
13.Add 50 µl of Stop solution into each well and mix thoroughly. The color changes into yellow immediately.
14. Read the O.D. absorbance at 450 nm in a microplate reader immediately after adding the stop solution.

Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit components

96 Assays

Storage

ELISA Microplate(Dismountable)8×12 strips4°C for 6 months
Lyophilized Standard24°C/-20°C
Sample/Standard Dilution Buffer20ml4°C
Biotin-labeled Antibody(Concentrated)120ul4°C (Protect from light)
Antibody Dilution Buffer10ml4°C
HRP-Streptavidin Conjugate(SABC)120ul4°C (Protect from light)
SABC Dilution Buffer10ml4°C
TMB Substrate10ml4°C (Protect from light)
Stop Solution10ml4°C
Wash Buffer(25X)30ml4°C
Plate Sealer5 -

Other materials and equipment required:

The ELISA Genie Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit will require other equipment and materials to carry out the assay. Please see list below for further details.
  • 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

Sample Preparation

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 TypeProtocol

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.

Human LRRK2 / Leucine-rich repeat serine / threonine-protein kinase 2 ELISA Kit Protein Information

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.
UniProt Protein Details:

Protein type:Protein kinase, TKL; Kinase, protein; Protein kinase, Ser/Thr (non-receptor); EC 2.7.11.1; TKL group; LRRK family

Chromosomal Location of Human Ortholog: 12q12

Cellular Component: axon; caveola; cell junction; cell soma; cytoplasm; cytoplasmic vesicle; cytosol; dendrite; dendrite cytoplasm; endoplasmic reticulum; endosome; extracellular space; Golgi apparatus; Golgi-associated vesicle; growth cone; inclusion body; intracellular; lysosome; microvillus; mitochondrial inner membrane; mitochondrial matrix; mitochondrial membrane; mitochondrial outer membrane; mitochondrion; neuron projection; perikaryon; plasma membrane; ribonucleoprotein complex; synaptic vesicle membrane; terminal button; trans-Golgi network

Molecular Function:actin binding; ATP binding; clathrin binding; glycoprotein binding; GTP binding; GTP-dependent protein kinase activity; GTPase activator activity; GTPase activity; identical protein binding; kinase activity; MAP kinase kinase activity; microtubule binding; protein binding; protein homodimerization activity; protein kinase A binding; protein kinase activity; protein serine/threonine kinase activity; receptor signaling complex scaffold activity; Rho GTPase binding; SNARE binding; syntaxin-1 binding; tubulin binding

Biological Process: activation of MAPK activity; activation of MAPKK activity; autophagy; calcium-mediated signaling; cellular response to starvation; determination of adult life span; endocytosis; Golgi organization and biogenesis; GTP metabolic process; intracellular distribution of mitochondria; lysosome organization and biogenesis; MAPKKK cascade; mitochondrion localization; mitochondrion organization and biogenesis; negative regulation of macroautophagy; negative regulation of protein amino acid phosphorylation; negative regulation of protein binding; neurite morphogenesis; neuromuscular junction development; olfactory bulb development; peptidyl-serine phosphorylation; peptidyl-threonine phosphorylation; phosphorylation; positive regulation of autophagy; positive regulation of dopamine receptor signaling pathway; positive regulation of GTPase activity; positive regulation of MAP kinase activity; positive regulation of programmed cell death; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; positive regulation of protein amino acid phosphorylation; positive regulation of protein binding; positive regulation of protein ubiquitination; protein amino acid autophosphorylation; protein amino acid phosphorylation; regulation of autophagy; regulation of dopamine receptor signaling pathway; regulation of excitatory postsynaptic membrane potential; regulation of locomotion; regulation of membrane potential; regulation of mitochondrial depolarization; regulation of neuron maturation; regulation of synaptic transmission, glutamatergic; response to oxidative stress; small GTPase mediated signal transduction; tangential migration from the subventricular zone to the olfactory bulb; Wnt receptor signaling pathway through beta-catenin

Disease: Parkinson Disease 8, Autosomal Dominant

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]
UniProt Code:Q5S007
NCBI GenInfo Identifier:294862450
NCBI Gene ID:120892
NCBI Accession:Q5S007.2
UniProt Secondary Accession:Q5S007,Q6ZS50, Q8NCX9, A6NJU2,
UniProt Related Accession:Q5S007
Molecular Weight:286,103 Da
NCBI Full Name:Leucine-rich repeat serine/threonine-protein kinase 2
NCBI Synonym Full Names:leucine-rich repeat kinase 2
NCBI Official Symbol:LRRK2  
NCBI Official Synonym Symbols:PARK8; RIPK7; ROCO2; AURA17; DARDARIN  
NCBI Protein Information:leucine-rich repeat serine/threonine-protein kinase 2
UniProt Protein Name:Leucine-rich repeat serine/threonine-protein kinase 2
UniProt Synonym Protein Names:Dardarin
Protein Family:Leucine-rich repeat serine/threonine-protein kinase
UniProt Gene Name:LRRK2  
UniProt Entry Name:LRRK2_HUMAN
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Additional Information

Product type:
ELISA
Reactivity:
Human
ELISA Type:
Sandwich
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