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[求助]
帮忙翻译一段实验操作步骤，条理清楚点就最好不过啦，万分感激
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这是我实验的一部分，自己翻译的总感觉不对，拜托各位大侠帮忙翻译一下，只要写出每个步骤怎么做就可以了，谢谢谢谢 2.2. Determination of total-SH, free-SH and S–S groups The amount of different sulphydryl group species was determined in products with a modified Ellman’s reaction (Bu¨ rki, 1977). Ellman’s reagent was prepared by dissolving 10 mg 5,50-dithio-bis(2-nitrobenzoic acid), DTNB, in 10mL 0.05M Tris/0.01 M EDTA-buffer. The pH was adjusted with 4M HCl to reach a pH of 7.0. The solution was degassed by ultrasound. Free sulfhydryl groups were determined by mixing 0.5mL milk with 2.5mL of 0.1M borate/0.02M EDTA, pH 8.3 buffer and 50 mL DTNBreagent in a 1 cm cell. After 20 min, the extinction at 412nm was measured. After addition of one drop of 30% H2O2, the extinction at 412nm was measured again; this gives the blank value. Concentration of free-sulphydryl groups in the sample was calculated using a calibration curve with cysteine. Total sulphydryl groups was determined by mixing 0.5mL milk with 2.5mL 0.05M borate/0.010M EDTA, pH 8.3/8.33M urea buffer and 50 mL DTNB-reagent in a 1 cm cell. Extinction at 412 nm was measured after 15 min, and the blank as above, after adding one drop of 30% H2O2. The concentration of total sulphydryl groups was calculated from a calibration curve with bovine serum albumin, which contains one buried sulphydryl group in its three-dimensional structure that is denatured by addition of urea in the buffer. The concentration of masked sulphydryl groups (MSH) was calculated by subtracting the concentration of free sulphydryl groups (FSH) from the concentration of total sulphydryl groups (TSH). The concentration of total protein-bound cysteine was determined by incubating 0.25mL milk with 1.25mL 0.1M Tris–HCl/12mM EDTA/0.6% SDS, pH 7.6/8 M ureum/ 10mM DTT buffer at 35 1C for 1 h. Subsequently, reduced protein and DTT were separated by eluting 0.25mL mixture on a GPC-column, using 75mM Tris/10mM EDTA/0.45% SDS, pH 7.5/6M urea buffer. After washing with 1.25mL buffer, 3mL elution fluid was collected. The collected fluid was mixed with 0.1mL 0.5M Tris-buffer and 50 mL DTNB-reagent. The extinction at 412 nm was measured between 15 and 90 min after addition of DTNB-reagent. The blank was again measured after addition of one drop of 30% H2O2. The concentration of S–S bridge was calculated by subtracting the concentration of total sulphydryl groups from the sulphydryl concentration after reduction, followed by a division by 2. |
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cmds1907
铁杆木虫 (著名写手)
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★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★ ★
天空shm: 金币+30, 翻译EPI+1, ★★★★★最佳答案, 太有用了，步骤说的很清晰 2014-08-21 08:53:56
天空shm: 金币+30, 翻译EPI+1, ★★★★★最佳答案, 太有用了，步骤说的很清晰 2014-08-21 08:53:56
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现上传译文的后两段： 滴加一滴30% 过氧化氢后, 再次测量412 nm处的消光系数，并得出空白值。样品中自由巯基基团的浓度采用与半胱氨酸校准曲线来计算。总巯基基团按如下法确定：将0.5 mL牛奶与2.5 mL 0.05 M硼酸盐/0.01M EDTA，pH8.3/8.33 M尿素缓冲溶液和50 mL DTNB-试剂混合，取适量于1cm 池长的比色皿中。15分钟后，测量412纳米处的消光系数，并同上滴加一滴30% 过氧化氢后测量空白值。总巯基基团的浓度可采用与牛血清白蛋白的校准曲线来计算，在其三维结构中包含一个隐藏的巯基基团，在缓冲溶液中加入尿素可（使其）变性。 掩蔽（隐藏）的巯基基团(MSH)的浓度可如下计算：总巯基基团(TSH)的浓度减去自由巯基基团(FSH)的浓度。与蛋白质相结合的半胱氨酸的总浓度可如下法确定：在35 摄氏度1小时内，配制（培养）0.25 mL牛奶1.25mL 0.1M Tris-HCl/12 mM EDTA/0.6% SDS，pH7.6/8 M 尿素/10 Mm DTT缓冲溶液；随后，被还原的蛋白质和DTT在一个GPC（凝胶渗透）-色谱柱上以0.25 mL 混合液洗脱分离，使用75 mM Tris/10 mM EDTA/0.45% SDS，pH7.5/6 M尿素缓冲溶液。用1.25 mL缓冲溶液洗涤后，收集3mL洗脱液。收集的液体与0.1 mL 0.5 M Tris 缓冲溶液和50 mL DTNB 试剂混合。在加入DTNB 试剂15至90分钟后，测量412纳米处的消光系数。滴加一滴30% 过氧化氢后再次测量空白值。S-S桥的浓度可如下计算：从还原后巯基的浓度减去总巯基基团的浓度，然后除以2。 |
4楼2014-08-20 18:10:23
cmds1907
铁杆木虫 (著名写手)
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【答案】应助回帖
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RXMCDM: 金币+1, 多谢应助！ 2014-08-20 16:57:08
RXMCDM: 金币+1, 多谢应助！ 2014-08-20 16:57:08
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先上传翻译的第一段，随后上传后两段。 2.2. 总SH，游离(自由)SH和S-S基团的测定 采用改进的Ellman反应(Bu¨ rki, 1977) 确定了产物中不同巯基（基团）物种的数量。Ellman试剂按如下法制备：10 mg 5,5-二硫代-双(2-硝基苯甲酸)，DTNB，溶解于10 mL 0.05 M Tris（注释）/ 0.01M EDTA 缓冲溶液中。用4 M盐酸调整pH值至7.0。溶液超声脱气。自由巯基基团按如下法确定：将0.5mL牛奶与2.5mL 0.1M硼酸盐/0.02M EDTA、pH 8.3缓冲溶液和50mL DTNB试剂混合，（取适量，否则逻辑上不通顺）于1cm 池长的比色皿中。20分钟后，测量412纳米处的消光系数。 Tris可能是三异丙基乙磺酰或三羟甲基氨基甲烷，这要根据实验部分中的“仪器及试剂”或其结构式来确认。 |
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天空shm2楼2014-08-20 16:44:45
3楼2014-08-20 17:32:44
