人間 HSD3B1 ELISA Kit (HUFI01896)- 高感度

商品名:	Human HSD3B1 / 3 beta-hydroxysteroid dehydrogenase ELISA Kit
製品コード:	HUFI01896
サイズ:	96T
エイリアス:	HSD3B1, 3 beta-hydroxysteroid dehydrogenase, Delta 5-->4-isomerase type I, 3-beta-HSD I, Trophoblast antigen FDO161G, 3BH, HSDB3A
検出方法:	Competitive ELISA, Coated with Antibody
申し込み:	This immunoassay kit allows for the in vitro quantitative determination of Human HSD3B1 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 Human HSD3B1 and the recovery rates were calculated by comparing the measured value to the expected amount of Human HSD3B1 in samples.

Matrix	Recovery range(%)	Average(%)
serum(n=5)	89-98	96
EDTA plasma(n=5)	85-102	94
UFH plasma(n=5)	89-101	95

直線性:

The linearity of the kit was assayed by testing samples spiked with appropriate concentration of Human HSD3B1 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)	89-101%	89-104%	89-103%
EDTA plasma(n=5)	83-98%	90-98%	83-93%
UFH plasma(n=5)	82-97%	85-98%	90-98%

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)	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 pipettetips
Incubator
Deionized or distilled water
Absorbent paper
Buffer resevoir

Uniprot

P14060

UniProt Protein Function:	HSD3B1: 3-beta-HSD is a bifunctional enzyme, that catalyzes the oxidative conversion of Delta(5)-ene-3-beta-hydroxy steroid, and the oxidative conversion of ketosteroids. The 3-beta-HSD enzymatic system plays a crucial role in the biosynthesis of all classes of hormonal steroids. Efficiently catalyzes the transformation of pregnenolone to progesterone, 17-alpha-hydroxypregnenolone to 17- alpha-hydroxyprogesterone, DHEA to 4-androstenedione, dihydrotestosterone to 5-alpha-androstane-3 beta,17 beta-diol, dehydroepiandrosterone to androstenedione and 5-alpha-androstan-3 beta,17 beta-diol to 5-alpha-dihydrotestosterone. Belongs to the 3-beta-HSD family.
UniProt Protein Details:	Protein type:Lipid Metabolism - androgen and estrogen; EC 5.3.3.1; Membrane protein, integral; EC 1.1.1.145; Oxidoreductase; Lipid Metabolism - C21-steroid hormone; Isomerase; Mitochondrial Chromosomal Location of Human Ortholog: 1p13.1 Cellular Component: endoplasmic reticulum membrane; mitochondrial inner membrane; mitochondrial intermembrane space; smooth endoplasmic reticulum membrane Molecular Function:3-beta-hydroxy-delta5-steroid dehydrogenase activity; steroid delta-isomerase activity Biological Process: androgen biosynthetic process; estrogen biosynthetic process; glucocorticoid biosynthetic process; mineralocorticoid biosynthetic process; steroid biosynthetic process
NCBI Summary:	The protein encoded by this gene is an enzyme that catalyzes the oxidative conversion of delta-5-3-beta-hydroxysteroid precursors into delta-4-ketosteroids, which leads to the production of all classes of steroid hormones. The encoded protein also catalyzes the interconversion of 3-beta-hydroxy- and 3-keto-5-alpha-androstane steroids. [provided by RefSeq, Jun 2016]
UniProt Code:	P14060
NCBI GenInfo Identifier:	112767
NCBI Gene ID:	3283
NCBI Accession:	P14060.2
UniProt Secondary Accession:	P14060,Q14545, Q8IV65, A8K691,
UniProt Related Accession:	P14060
Molecular Weight:	42,252 Da
NCBI Full Name:	3 beta-hydroxysteroid dehydrogenase/Delta 5-->
NCBI Synonym Full Names:	hydroxy-delta-5-steroid dehydrogenase, 3 beta- and steroid delta-isomerase 1
NCBI Official Symbol:	HSD3B1
NCBI Official Synonym Symbols:	I; HSD3B; HSDB3; HSDB3A; SDR11E1; 3BETAHSD
NCBI Protein Information:	3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type 1
UniProt Protein Name:	3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type 1
UniProt Synonym Protein Names:	3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase type I; 3-beta-HSD I; Trophoblast antigen FDO161GIncluding the following 2 domains:3-beta-hydroxy-Delta(5)-steroid dehydrogenase (EC:1.1.1.145)Alternative name(s):3-beta-hydroxy-5-ene steroid dehydrogenase; Progesterone reductase
Protein Family:	3 beta-hydroxysteroid dehydrogenase/Delta 5-->4-isomerase
UniProt Gene Name:	HSD3B1
UniProt Entry Name:	3BHS1_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。

使用前に、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-coatedplate respectively, and then, record their positions. It isrecommended to measure each standard and sample in duplicate. Washplate 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 blankwell is added with Sample Dilution Buffer. Immediately add 50 µL of biotin-labelled antibody workingsolution to each well. Cover with the plate sealer provided. Gently tap the plate to ensure thoroughmixing. 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 timesWash 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 essentialto good performance. After the last wash, remove any remaining WashBuffer by aspirating or decanting. Invert the plate and pat it againstthick clean absorbent paper.
4.	HRP-Streptavidin Conjugate(SABC): Add 100µL of SABC workingsolution to each well. Cover with a new Plate sealer. Incubate for30minutes at 37°C.
5.	Wash: Repeat the aspiration/wash process for five times.
6.	TMB Substrate: Add 90µL of TMB Substrate to each well. Coverwith a new Plate sealer. Incubate for about 10-20 minutes at 37°C.Protect from light. The reaction time can be shortened or extendedaccording to the actual color change, but not more than 30minutes.When apparent gradient appeared in standard wells, you can terminatethe reaction.
7.	Stop: Add 50µL of Stop Solution to each well. Color turn toyellow immediately. The adding order of stop solution should be as thesame as the substrate solution.
8.	OD Measurement: Determine the optical density (OD Value) of each wellat once, using a microplate reader set to 450 nm. You should open themicroplate 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.