ねずみ CRH ELISA (MOFI00297)

商品名:	Mouse CRH (Corticotropin Releasing Hormone) ELISA Kit
製品コード:	MOFI00297
サイズ:	96 Assays
エイリアス:	Crh, Corticotropin Releasing Hormone, Corticoliberin, Corticotropin-releasing factor, CRF, Corticotropin-releasing hormone
検出方法:	Competitive ELISA
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Mouse Crh concentrations in serum plasma and other biological fluids.
感度:	0.188ng/ml
範囲:	0.313-20ng/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 Crh and the recovery rates were calculated by comparing the measured value to the expected amount of Mouse Crh in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	85-98	93
EDTA plasma(n=5)	89-99	94
UFH plasma(n=5)	86-100	96

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Mouse Crh 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)	85-105%	88-103%	94-102%
EDTA plasma(n=5)	87-97%	82-101%	87-98%
UFH plasma(n=5)	80-98%	80-99%	83-100%

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)	60ul	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

Q8CIT0

UniProt Protein Function:	CRH: This hormone from hypothalamus regulates the release of corticotropin from pituitary gland. Belongs to the sauvagine/corticotropin-releasing factor/urotensin I family.
UniProt Protein Details:	Protein type:Secreted; Secreted, signal peptide Cellular Component: cell soma; cytoplasm; extracellular region; extracellular space; perikaryon Molecular Function:corticotropin-releasing hormone receptor 1 binding; corticotropin-releasing hormone receptor 2 binding; hormone activity Biological Process: adrenal gland development; associative learning; female pregnancy; glucocorticoid biosynthetic process; induction of apoptosis by hormones; inflammatory response; learning and/or memory; lung development; negative regulation of blood pressure; negative regulation of epinephrine secretion; negative regulation of luteinizing hormone secretion; positive regulation of adrenocorticotropic hormone secretion; positive regulation of cAMP biosynthetic process; positive regulation of cell proliferation; positive regulation of cortisol secretion; positive regulation of protein amino acid phosphorylation; regulation of serotonin secretion; synaptic transmission, dopaminergic
NCBI Summary:	This gene encodes a member of the corticotropin-releasing factor family and preproprotein that is proteolytically processed to generate a mature protein product. This protein product is a neuropeptide hormone that binds to the corticotropin releasing hormone receptors (CRHR1 and CRHR2) to stimulate the release of adrenocorticotropic hormone from the pituitary gland in response to stress. The encoded protein may also regulate angiogenesis and inflammation. Homozygous knockout mice for this gene exhibit reduced corticosterone levels while the offspring of these mice die perinatally. [provided by RefSeq, Aug 2015]
UniProt Code:	Q8CIT0
NCBI GenInfo Identifier:	45429990
NCBI Gene ID:	12918
NCBI Accession:	NP_991338.1
UniProt Secondary Accession:	Q8CIT0,Q14AA2,
UniProt Related Accession:	Q8CIT0
Molecular Weight:	20,778 Da
NCBI Full Name:	corticoliberin preproprotein
NCBI Synonym Full Names:	corticotropin releasing hormone
NCBI Official Symbol:	Crh
NCBI Official Synonym Symbols:	CRF; Gm1347
NCBI Protein Information:	corticoliberin
UniProt Protein Name:	Corticoliberin
UniProt Synonym Protein Names:	Corticotropin-releasing factor; CRF; Corticotropin-releasing hormone
Protein Family:	Corticoliberin
UniProt Gene Name:	Crh
UniProt Entry Name:	CRF_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.	Add Sample and Biotin-detection antibody: Add 50µL of Standard, Blank or Sample per well. The blank well is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody working solution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thorough mixing. Incubate for 45 minutes at 37°C. (Solutions are added to the bottom of micro-ELISA platewell, avoid touching plate walls and foaming).
3.	Wash: Aspirate each well and wash, repeating the process three times. Wash by filling each well with Wash Buffer (approximately 350µL) using a squirt bottle, multi-channel pipette, manifold dispenser orautomated washer. Complete removal of liquid at each step is essential to good performance. After the last wash, remove any remaining Wash Buffer by aspirating or decanting. Invert the plate and pat it against thick clean absorbent paper.
4.	HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC working solution to each well. Cover with a new Plate sealer. Incubate for 30 minutes at 37°C.
5.	Wash: Repeat the aspiration/wash process for five times.
6.	TMB Substrate: Add 90µL of TMB Substrate to each well. Cover with a new Plate sealer. Incubate for about 10-20 minutes at 37°C. Protect from light. The reaction time can be shortened or extended according to the actual color change, but not more than 30minutes. When apparent gradient appeared in standard wells, you can terminate the reaction.
7.	Stop: Add 50µL of Stop Solution to each well. Color turn to yellow immediately. The adding order of stop solution should be as the same as the substrate solution.
8.	OD Measurement: Determine the optical density (OD Value) of each well at once, using a microplate reader set to 450 nm. You should open the microplate reader ahead, preheat the instrument, and set the testing parameters.

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.