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yxzhang838

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α-葡萄糖苷酶抑制活性的测定100 μL反应体系中适量哺乳动物来源的葡萄糖苷酶（α-glucosidase-rat，由大鼠小肠组织中提取）、67 nM磷酸钠缓冲液(pH 6.8)和样品，同时设立空白对照(不含酶和样品)和阴性对照(不含样品)，37℃反应10min，加入0.1 M麦芽糖，室温反应10 min，再加入200 μL的葡萄糖检测试剂，混匀后490 nM测定OD值。根据OD值计算抑制率，抑制率=[1-(OD样品-OD空白)/(OD阴性-OD空白)]×100。每个样品每个浓度设双复孔，重复两次。

PPAR反应元件(PPRE)激动活性检测试验HepG2肝癌细胞常规培养于37℃、5%CO2、含100U•mL-1链霉素和青霉素的低糖DMEM中。1.5×104个/孔接种于96孔板后培养过夜，参照转染试剂说明书进行质粒转染。转染的质粒包括带有PPAR反应元件和萤火虫荧光素酶报告基因的质粒pPPRE-Luc，及用作转染内参照的带有海肾荧光素酶的质粒phRL-TK，转染24h后换用含待测样品的培养基，同时设立空白对照(未转染的细胞)、阴性对照(转染的细胞不加样品)和阳性对照(转染的细胞加入匹格列酮，Pioglitazone)。继续培养24h后用双荧光素酶报告基因检测试剂盒(Promega)检测荧光素酶活性，根据检测到的化学发光强度L值计算激动率。L1为萤火虫荧光素酶的化学发光强度，L2为内参照海肾荧光素酶的化学发光强度，激动率=[(L1样品-L1空白)/(L1阴性-L1空白)]/[(L2样品-L2空白)/(L2阴性-L2空白)]×100%；样品检测浓度为10μg•mL-1，检测时设双复孔，重复两次。

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1楼 2009-09-26 13:27:28

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goodtimega

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α-glucosidase inhibitory activity is determined through 100 μL reaction system suitable mammalian source glucosidase (α-glucosidase-rat that was extracted from the rat small intestine)with 67 nM sodium phosphate buffer (pH 6.8). This sets up a blank control (without enzyme and samples) and negative control (without sample) through 37 ℃ reaction to 10min, adding 0.1 M maltose, after room temperature reaction of 10 min, then adding 200 μL of glucose testing reagents, mixing post-490 nM measured OD values. Inhibition rate calculated is in accordance to OD values of the inhibition rate = [1 - (OD sample-OD blank) / (OD negative-OD blank)] × 100. The concentration of each sample shows a double recovery for each hole after being repeated twice.

PPAR response element (PPRE) activity in emotion-testing of conventional HepG2 hepatoma cells was cultured in 37 ℃, 5% CO2, containing 100U • mL-1 streptomycin and penicillin in the sugar DMEM. 1.5 × 104 / holes were inoculated into 96-well plates cultured overnight, taking into account the instructions for plasmid transfection reagent transfection. A plasmid was transferred including the PPAR-responsive element and the firefly luciferase reporter gene plasmid pPPRE-Luc, and being used as a reference within transfected with a Renilla luciferase plasmid phRL-TK, and transfection 24h. After changing the medium containing the sample under test, the establishment of blank control (non-transfected cells), negative control (cells transfected without samples), and positive control (cells transfected by adding Pioglitazone, Pioglitazone) was achived. This process was continued to foster 24h later Dual-Luciferase Reporter Gene Assay Kit (Promega) through detection of luciferase activity, according to detected values of chemiluminescence intensity of L calculated the rate of excited. L1 stands for the firefly luciferase chemiluminescence intensity, L2 as an internal reference Renilla luciferase chemiluminescence intensity, excitement rate = [(L1 Sample-L1 blank) / (L1-negative-L1 blank )]/[( L2 Sample -L2 blank) / (L2-negative-L2 blank)] × 100%. Samples detected concentrations of 10μg • mL-1, a double re-hole detection were repeated twice.

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2楼2009-09-27 02:18:48

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zhuzhaofu

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alpha - In the grape glycosidase suppression active determination 100 μL reacting system the right amount mammal originates the grape glycosidase (α-glucosidase-rat, withdraws, 67 nM sodium phosphate buffer solution by big mouse small intestine organization in) (pH 6.8) and the sample, simultaneously sets up the blank comparison (not including enzyme and sample) and the negative comparison (not including sample), 37℃ responds 10min, joins 0.1 M malt sugar, the room temperature responded 10 min, joins 200 μL glucose examination reagent again, mixes uniform latter 490 nM to determine the OD value.According to OD value computation suppression rate, suppression rate = [1- (OD sample - OD blank)/(OD negative - OD blank)]×100.Each sample each density supposes the double duplicate hole, duplicates two times.
PPAR responded the part (PPRE) excited activeness examination experiments the HepG2 liver cancer cell convention raise in 37℃, 5%CO2, contains 100U•mL-1 chain mildew element and in penicillin low sugar DMEM.1.5×104 a/vaccination raises the over night after 96 orifices, the reference extension dyes the reagent instruction booklet to carry on the material particle extension to dye.The extension dyes the material particle including has the PPAR response part and firefly fluorescein enzyme report gene material particle pPPRE-Luc, and serves as the extension to dye the materials for internal reference to illuminate has sea kidney fluorescein enzyme material particle phRL-TK, after the extension dyes 24h to use in exchange including treats measured (cell which the sample the culture medium, simultaneously sets up (which the blank comparison not to have transferred dyes cell), the negative comparison extension dyes not to add (cell which sample) and masculine comparison extension dyes to join the Geleg alkone, Pioglitazone).After continues to raise 24h with the double fluorescein enzyme report gene examination reagent box (Promega) examination fluorescein enzyme activity, according to chemiluminescence intensity L value computation which examines excited rate.L1 is the firefly fluorescein enzyme chemiluminescence intensity, L2 is the materials for internal reference according to the sea kidney fluorescein enzyme chemiluminescence intensity, excited rate = [(L1 sample - L1 blank)/(L1 negative - L1 blank)]/[(L2 sample - L2 blank)/(L2 negative - L2 blank)]×100%; Sample examination density for 10μg•mL-1, when examination supposes the double duplicate hole, duplicates two times.
只是在网上翻译的，看看能不能帮楼主！支持！

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3楼2009-09-27 09:10:48

已阅关注TA 给TA发消息送TA红花 TA的回帖

yxzhang838

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引用回帖:

Originally posted by goodtimega at 2009-9-27 02:18:
α-glucosidase inhibitory activity is determined through 100 μL reaction system suitable mammalian source glucosidase (α-glucosidase-rat that was extracted from the rat small intestine)with 67 nM ...

我怎末赠送你金币呢？
你第二段是不是在线翻译啊？