荧光定量PCR仪在蛋白结晶条件筛选方面的应用
本帖最后由 浙农林—老张 于 2012-12-12 11:26 编辑static/image/filetype/pdf.gif
Differential Scanning Fluorimetry Measurement of Protein Stability Changes upon .pdf(263.09 KB, 下载次数: 9)2012-12-12 10:56 上传点击文件名下载附件荧光定量PCR仪可应用于蛋白热稳定性测试实验，使用的染料SYPRO。ABI7500/BIORAD IQ5及其以上机型都可以做。实验方便快捷，1-2小时搞定，还可以高通量筛选拟结晶的不同条件下蛋白的稳定性。从经验上而言，蛋白热稳定性越好，稳定性就高，也就容易结晶。
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Thermofluor-based optimization strategy for the stabilization and crystallizatio.pdf(904.81 KB, 下载次数: 8)2012-12-12 10:56 上传点击文件名下载附件操作很简单，过程很轻松。哈哈 Differential scanning fluorimetryThe melting temperature of N. meningitidis DHDPS and E. coli DHDPS were determined by differential scanning fluorimetry using a minor variation on the method of Nordlund et al.
. Briefly, the protein samples (4 μL) were added with mixing to buffer (20 μL) and SYPRO orange dye (1 μL, diluted 20-fold from supplied concentration with dH2O) in a 96 well microplate. After sealing the plate, a stepped thermal melt program starting at 20 °C and finishing at 95 °C in 0.3 °C increments, with a dwell time of 20 s after each temperature rise, was run on an IQ5 Bio-Rad iCycler iQ5 Multicolour Real-Time PCR Detection System. Fluorescence measurements were obtained at the end of each dwell time. The reading of a blank well containing buffer and dye but lacking protein was subtracted from each sample reading. Melting temperatures were calculated as the temperature of maximum inflection of the melting curve. Samples were measured in triplicate.
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ClassicTech2.ppt(141 KB, 下载次数: 3)2012-12-17 16:04 上传点击文件名下载附件阅读权限: 10原理PPT 有点意思，看过用荧光定量PCR仪测含FAD辅酶蛋白的Tm值的文章，新应用可以了解下
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