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以东农冬麦1号为材料,对苗期地下茎处的蛋白提取方法、蛋白溶解、上样量、胶条的转移等方面进行了试验。结果表明:在蛋白提取方面,TCA/丙酮法(T法)和尿素/硫脲法(N法)相比T法能减少低丰度蛋白的损失得到蛋白点数更多的图谱。在蛋白溶解方面,经过两次水化液溶解的蛋纯度较高,在等电聚焦时能保持8000伏较高电压。上样量方面,10mg粗蛋白溶于两次水化液能得到清晰、分离效果好、蛋白点数较多的图像。胶条转移方面,先向胶面中加入400μl 0.3%普通琼脂糖溶液后,用200μl的电极缓冲液冲洗胶条支撑膜会使胶条顺利转移到第二向胶面上且胶条与胶面间不会产生气泡。
Taking Dongnong Dongmai 1 as material, the methods of protein extraction, protein lysis, sample loading, tape transfer and so on were studied. The results showed that TCA / acetone method (T method) and urea / thiourea method (N method) could reduce the abundance of low abundance protein and get more protein spots compared with T method in protein extraction. In terms of protein lysis, the egg dissolved in two hydration solutions has a higher purity and maintains a higher voltage of 8000 V during isoelectric focusing. The amount of sample, 10mg crude protein dissolved in two hydration can be clear, good separation, protein spots more images. Strip transfer, first to the plastic surface by adding 400μl 0.3% ordinary agarose solution, with 200μl of electrode buffer solution strip will strip the adhesive successfully transferred to the second side of the rubber surface and the strip and the plastic surface Will not produce bubbles between.