ねずみ AGER/RAGE ELISA (MOFI00350)

商品名:	Mouse AGER / RAGE ELISA Kit
製品コード:	MOFI00350
サイズ:	96 Assays
エイリアス:	RAGE, AGER
検出方法:	Sandwich ELISA
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Mouse AGER concentrations in serum plasma and other biological fluids.
感度:	75pg/ml
範囲:	125-8000pg/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 AGER and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse AGER in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	86-101	92
EDTA plasma(n=5)	89-103	95
UFH plasma(n=5)	89-98	93

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse AGER 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)	88-95%	90-105%	87-97%
EDTA plasma(n=5)	83-99%	83-101%	83-100%
UFH plasma(n=5)	83-95%	83-93%	80-100%

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

Q62151

UniProt Protein Function:	RAGE: Mediates interactions of advanced glycosylation end products (AGE). These are nonenzymatically glycosylated proteins which accumulate in vascular tissue in aging and at an accelerated rate in diabetes. Acts as a mediator of both acute and chronic vascular inflammation in conditions such as atherosclerosis and in particular as a complication of diabetes. AGE/RAGE signaling plays an important role in regulating the production/expression of TNF- alpha, oxidative stress, and endothelial dysfunction in type 2 diabetes. Interaction with S100A12 on endothelium, mononuclear phagocytes, and lymphocytes triggers cellular activation, with generation of key proinflammatory mediators. Interaction with S100B after myocardial infarction may play a role in myocyte apoptosis by activating ERK1/2 and p53/TP53 signaling. Receptor for amyloid beta peptide. Contributes to the translocation of amyloid-beta peptide (ABPP) across the cell membrane from the extracellular to the intracellular space in cortical neurons. ABPP-initiated RAGE signaling, especially stimulation of p38 mitogen-activated protein kinase (MAPK), has the capacity to drive a transport system delivering ABPP as a complex with RAGE to the intraneuronal space. Interacts with S100B, S100A1 and APP. Interacts with S100A12. Endothelial cells. 4 isoforms of the human protein are produced by alternative splicing.
UniProt Protein Details:	Protein type:Receptor, misc.; Motility/polarity/chemotaxis; Membrane protein, integral; Cell cycle regulation Cellular Component: extracellular space; cell surface; cell soma; membrane; axon; cytoplasm; plasma membrane; integral to membrane; basal plasma membrane; cytosol Molecular Function:identical protein binding; protein binding; advanced glycation end-product receptor activity Biological Process: positive regulation of JNK activity; positive regulation of apoptosis; negative regulation of collagen biosynthetic process; positive regulation of smooth muscle cell proliferation; negative regulation of cell adhesion; JAK-STAT cascade; positive regulation of smooth muscle cell migration; induction of positive chemotaxis; activation of NF-kappaB transcription factor; calcium ion homeostasis; positive regulation of fibroblast proliferation; negative regulation of protein amino acid phosphorylation; regulation of DNA binding; positive regulation of neuron apoptosis; regulation of inflammatory response; negative regulation of endothelial cell proliferation; response to hypoxia; positive regulation of protein amino acid phosphorylation; inflammatory response; positive regulation of phagocytosis, engulfment; neurite development; positive regulation of autophagy; positive regulation of cell migration; positive regulation of inflammatory response
UniProt Code:	Q62151
NCBI GenInfo Identifier:	2497318
NCBI Gene ID:	11596
NCBI Accession:	Q62151.1
UniProt Related Accession:	Q62151
Molecular Weight:	42,669 Da
NCBI Full Name:	Advanced glycosylation end product-specific receptor
NCBI Synonym Full Names:	advanced glycosylation end product-specific receptor
NCBI Official Symbol:	Ager
NCBI Official Synonym Symbols:	RAGE
NCBI Protein Information:	advanced glycosylation end product-specific receptor; advanced glycation end-products receptor; receptor for advanced glycosylation end products; receptor for advanced glycation end-products variant 20
UniProt Protein Name:	Advanced glycosylation end product-specific receptor
UniProt Synonym Protein Names:	Receptor for advanced glycosylation end products
Protein Family:	Advanced glycosylation end product-specific receptor
UniProt Gene Name:	Ager
UniProt Entry Name:	RAGE_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.