人間 PARK2 ELISA Kit (HUFI03035)- 高感度

商品名:	Human PARK2(E3 ubiquitin-protein ligase parkin) ELISA Kit
製品コード:	HUFI03035
サイズ:	96T
エイリアス:	E3 ubiquitin-protein ligase parkin
検出方法:	Sandwich ELISA, Double Antibody
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Human PARK2 concentrations in serum plasma and other biological fluids.
感度:	0.094ng/ml
範囲:	0.156-10ng/ml
保管所:	4°C for 6 months
ノート:	For Research Use Only

回復:

Matrices listed below were spiked with certain level of Human PARK2 and the recovery rates were calculated by comparing the measured value to the expected amount of Human PARK2 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	88-103	97
EDTA plasma(n=5)	87-104	93
UFH plasma(n=5)	90-102	96

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human PARK2 and their serial dilutions. The results were demonstrated by the percentage of calculated concentration to the expected.

Sample	1:2	1:4	1:8
serum(n=5)	89-96%	88-102%	85-95%
EDTA plasma(n=5)	83-98%	93-101%	82-98%
UFH plasma(n=5)	84-93%	89-100%	84-99%

CV(%):

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

成分	量	保管所
ELISA Microplate (Dismountable)	8×12 strips	4°C for 6 months
Lyophilized Standard	2	4°C/-20°C
Sample/Standard Dilution Buffer	20ml	4°C
Biotin-labeled Antibody(Concentrated)	120ul	4°C (Protect from light)
Antibody Dilution Buffer	10ml	4°C
HRP-Streptavidin Conjugate(SABC)	120ul	4°C (Protect from light)
SABC Dilution Buffer	10ml	4°C
TMB Substrate	10ml	4°C (Protect from light)
Stop Solution	10ml	4°C
Wash Buffer(25X)	30ml	4°C
Plate Sealer	5	-

必要なその他の材料と設備:

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

Uniprot

O60260

UniProt Protein Function:	PARK2: a component of a multiprotein E3 ubiquitin ligase complex, catalyzing the covalent attachment of ubiquitin moieties onto substrate proteins, such as BCL2, SYT11, CCNE1, GPR37, STUB1, a 22 kDa O-linked glycosylated isoform of SNCAIP, SEPT5, ZNF746 and AIMP2. Mediates monoubiquitination as well as 'Lys-48'-linked and 'Lys-63'-linked polyubiquitination of substrates depending on the context. Participates in the removal and/or detoxification of abnormally folded or damaged protein by mediating 'Lys-63'-linked polyubiquitination of misfolded proteins such as PARK7: 'Lys-63'- linked polyubiquitinated misfolded proteins are then recognized by HDAC6, leading to their recruitment to aggresomes, followed by degradation. Mediates 'Lys-63'-linked polyubiquitination of SNCAIP, possibly playing a role in Lewy-body formation. Mediates monoubiquitination of BCL2, thereby acting as a positive regulator of autophagy. Promotes the autophagic degradation of dysfunctional depolarized mitochondria. Mediates 'Lys-48'-linked polyubiquitination of ZNF746, followed by degradation of ZNF746 by the proteasome; possibly playing a role in role in regulation of neuron death. Limits the production of reactive oxygen species (ROS). Loss of this ubiquitin ligase activity appears to be the mechanism underlying pathogenesis of PARK2. May protect neurons against alpha synuclein toxicity, proteasomal dysfunction, GPR37 accumulation, and kainate-induced excitotoxicity. May play a role in controlling neurotransmitter trafficking at the presynaptic terminal and in calcium-dependent exocytosis. Regulates cyclin-E during neuronal apoptosis. May represent a tumor suppressor gene. Forms an E3 ubiquitin ligase complex with UBE2L3 or UBE2L6. Mediates 'Lys-63'-linked polyubiquitination by associating with UBE2V1. Part of a SCF-like complex, consisting of PARK2, CUL1 and FBXW7. Part of a complex, including STUB1, HSP70 and GPR37. The amount of STUB1 in the complex increases during ER stress. STUB1 promotes the dissociation of HSP70 from PARK2 and GPR37, thus facilitating PARK2-mediated GPR37 ubiquitination. HSP70 transiently associates with unfolded GPR37 and inhibits the E3 activity of PARK2, whereas, STUB1 enhances the E3 activity of PARK2 through promotion of dissociation of HSP70 from PARK2-GPR37 complexes. Interacts with PSMD4 and PACRG. Interacts with LRRK2. Interacts with RANBP2. Interacts with SUMO1 but not SUMO2, which promotes nuclear localization and autoubiquitination. Interacts (via first RING- type domain) with AIMP2 (via N-terminus). Interacts with PSMA7 and RNF41. Interacts with PINK1. Highly expressed in the brain including the substantia nigra. Expressed in heart, testis and skeletal muscle. Expression is down-regulated or absent in tumor biopsies, and absent in the brain of PARK2 patients. Overexpression protects dopamine neurons from kainate-mediated apoptosis. Found in serum. Belongs to the RBR family. Parkin subfamily. 6 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Ubiquitin ligase; Ligase; Ubiquitin conjugating system; EC 6.3.2.19; EC 6.3.2.- Chromosomal Location of Human Ortholog: 6q25.2-q27 Cellular Component: Golgi apparatus; neuron projection; mitochondrion; perinuclear region of cytoplasm; endoplasmic reticulum; cytoplasm; SCF ubiquitin ligase complex; nucleus; cytosol; ubiquitin ligase complex Molecular Function:tubulin binding; identical protein binding; ubiquitin binding; zinc ion binding; histone deacetylase binding; ubiquitin-protein ligase activity; Hsp70 protein binding; actin binding; protein kinase binding; PDZ domain binding; protein binding; G-protein-coupled receptor binding; ubiquitin conjugating enzyme binding; chaperone binding; ubiquitin protein ligase binding; heat shock protein binding; kinase binding; SH3 domain binding; ligase activity Biological Process: protein monoubiquitination; proteasomal ubiquitin-dependent protein catabolic process; negative regulation of JNK cascade; negative regulation of actin filament bundle formation; startle response; central nervous system development; protein polyubiquitination; adult locomotory behavior; regulation of protein ubiquitination; protein ubiquitination during ubiquitin-dependent protein catabolic process; regulation of neurotransmitter secretion; protein ubiquitination; mitochondrion localization; norepinephrine metabolic process; dopamine metabolic process; regulation of dopamine secretion; negative regulation of insulin secretion; negative regulation of glucokinase activity; zinc ion homeostasis; negative regulation of protein amino acid phosphorylation; regulation of lipid transport; dopamine uptake; negative regulation of neuron apoptosis; positive regulation of DNA binding; mitochondrion organization and biogenesis; mitochondrial fission; synaptic transmission, glutamatergic; protein autoubiquitination; positive regulation of I-kappaB kinase/NF-kappaB cascade; transcription, DNA-dependent; protein stabilization; learning; regulation of protein transport; cellular protein catabolic process; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; mitochondrion degradation; positive regulation of transcription from RNA polymerase II promoter; regulation of autophagy; response to oxidative stress Disease: Parkinson Disease 2, Autosomal Recessive Juvenile; Leprosy, Susceptibility To, 2; Lung Cancer; Ovarian Cancer
NCBI Summary:	The precise function of this gene is unknown; however, the encoded protein is a component of a multiprotein E3 ubiquitin ligase complex that mediates the targeting of substrate proteins for proteasomal degradation. Mutations in this gene are known to cause Parkinson disease and autosomal recessive juvenile Parkinson disease. Alternative splicing of this gene produces multiple transcript variants encoding distinct isoforms. Additional splice variants of this gene have been described but currently lack transcript support. [provided by RefSeq, Jul 2008]
UniProt Code:	O60260
NCBI GenInfo Identifier:	116242725
NCBI Gene ID:	5071
NCBI Accession:	O60260.2
UniProt Secondary Accession:	O60260,Q5TFV8, Q5VVX4, Q6Q2I6, Q8NI41, Q8NI43, Q8NI44 Q8WW07, A3FG77, A8K975, D3JZW7, D3K2X0,
UniProt Related Accession:	O60260
Molecular Weight:	465
NCBI Full Name:	E3 ubiquitin-protein ligase parkin
NCBI Synonym Full Names:	parkin RBR E3 ubiquitin protein ligase
NCBI Official Symbol:	PARK2
NCBI Official Synonym Symbols:	PDJ; PRKN; AR-JP; LPRS2
NCBI Protein Information:	E3 ubiquitin-protein ligase parkin; parkinson juvenile disease protein 2; parkinson protein 2, E3 ubiquitin protein ligase (parkin); Parkinson disease (autosomal recessive, juvenile) 2, parkin
UniProt Protein Name:	E3 ubiquitin-protein ligase parkin
UniProt Synonym Protein Names:	Parkinson juvenile disease protein 2; Parkinson disease protein 2
Protein Family:	E3 ubiquitin-protein ligase
UniProt Gene Name:	PARK2
UniProt Entry Name:	PRKN2_HUMAN

*ノート: プロトコルは、各バッチ/ロットに固有です。正しい手順については、キットに含まれているプロトコルに従ってください。

ウェルに加える前に、SABCワーキング溶液とTMB基質を37°Cで少なくとも30分間平衡化します。サンプルと試薬を希釈するときは、完全に均一に混合する必要があります。各テストの標準曲線をプロットすることをお勧めします。

ステップ	プロトコル
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.

ELISAアッセイを実施する場合、可能な限り最良の結果を達成するためにサンプルを準備することが重要です。以下に、さまざまなサンプルタイプのサンプルを準備するための手順のリストを示します。

サンプルタイプ	プロトコル
血清	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.
プラズマ	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.
尿および脳脊髄液	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.
細胞培養上清	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.
細胞溶解物	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.
組織ホモジネート	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.
組織溶解物	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.
母乳	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.