ねずみ Clusterin ELISA (MOFI00026)

商品名:	Mouse Clusterin ELISA Kit
製品コード:	MOFI00026
サイズ:	96 Assays
エイリアス:	CLU, Clusterin, CLU, APO-J, APOJ, CLI, KUB1, NA1, NA2, SGP2, SP-40, TRPM2
検出方法:	Sandwich ELISA
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Mouse CLU concentrations in serum plasma and other biological fluids.
感度:	1.5ng/ml
範囲:	2.5-160ng/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 Mouse CLU and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse CLU in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-105	97
EDTA plasma(n=5)	96-105	100
UFH plasma(n=5)	88-103	93

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse CLU 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)	86-96%	95-104%	92-105%
EDTA plasma(n=5)	84-100%	88-99%	86-98%
UFH plasma(n=5)	81-97%	81-94%	81-90%

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

Q06890

UniProt Protein Function:	CLU: Isoform 1 functions as extracellular chaperone that prevents aggregation of nonnative proteins. Prevents stress- induced aggregation of blood plasma proteins. Inhibits formation of amyloid fibrils by APP, APOC2, B2M, CALCA, CSN3, SNCA and aggregation-prone LYZ variants (in vitro). Does not require ATP. Maintains partially unfolded proteins in a state appropriate for subsequent refolding by other chaperones, such as HSPA8/HSC70. Does not refold proteins by itself. Binding to cell surface receptors triggers internalization of the chaperone-client complex and subsequent lysosomal or proteasomal degradation. Secreted isoform 1 protects cells against apoptosis and against cytolysis by complement. Intracellular isoforms interact with ubiquitin and SCF (SKP1-CUL1-F-box protein) E3 ubiquitin-protein ligase complexes and promote the ubiquitination and subsequent proteasomal degradation of target proteins. Promotes proteasomal degradation of COMMD1 and IKBKB. Modulates NF-kappa-B transcriptional activity. Nuclear isoforms promote apoptosis. Mitochondrial isoforms suppress BAX-dependent release of cytochrome c into the cytoplasm and inhibit apoptosis. Plays a role in the regulation of cell proliferation. Belongs to the clusterin family. 5 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Secreted, signal peptide; Apoptosis; Secreted; Mitochondrial Cellular Component: extracellular matrix; extracellular space; neuron projection; growth cone; intracellular membrane-bound organelle; membrane; mitochondrion; endoplasmic reticulum; perinuclear region of cytoplasm; cytoplasm; extracellular region; cytoplasmic vesicle; nucleus Molecular Function:protein binding; ubiquitin protein ligase binding; protein N-terminus binding; misfolded protein binding Biological Process: cell death; response to misfolded protein; positive regulation of nitric oxide biosynthetic process; protein stabilization; positive regulation of apoptosis; cell morphogenesis; microglial cell activation; positive regulation of tumor necrosis factor production; endocrine pancreas development; activation of NF-kappaB transcription factor; negative regulation of protein homooligomerization; myelin maintenance in the central nervous system; positive regulation of proteasomal ubiquitin-dependent protein catabolic process; protein import; positive regulation of cell proliferation; chaperone-mediated protein complex assembly; positive regulation of cell differentiation; neurite morphogenesis; negative regulation of apoptosis
NCBI Summary:	The protein encoded by this gene is a secreted chaperone that can under some stress conditions also be found in the cell cytosol. It has been suggested to be involved in several basic biological events such as cell death, tumor progression, and neurodegenerative disorders. [provided by RefSeq, May 2011]
UniProt Code:	Q06890
NCBI GenInfo Identifier:	729152
NCBI Gene ID:	12759
NCBI Accession:	Q06890.1
UniProt Related Accession:	Q06890
Molecular Weight:	51,656 Da
NCBI Full Name:	Clusterin
NCBI Synonym Full Names:	clusterin
NCBI Official Symbol:	Clu
NCBI Official Synonym Symbols:	Cli; ApoJ; Sgp2; SP-40; Sgp-2; Sugp-2; AI893575; D14Ucla3
NCBI Protein Information:	clusterin; apo-J; clustrin; Apolipoprotein J; sulfated glycoprotein 2; complement lysis inhibitor; testosterone repressed prostate message; testosterone repressed prostate message-2
UniProt Protein Name:	Clusterin
UniProt Synonym Protein Names:	Apolipoprotein J; Apo-J; Clustrin; Sulfated glycoprotein 2; SGP-2
Protein Family:	Clusterin
UniProt Gene Name:	Clu
UniProt Entry Name:	CLUS_MOUSE

*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。

ステップ	手順
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.