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小扬仔

新虫 (小有名气)

应助: 3 (幼儿园)
金币: 246.6
帖子: 263
在线: 17.9小时
虫号: 3114770
注册: 2014-04-05
专业: 生物化学

[求助] 植物原生质体制备和吸水膨胀、磷酸化蛋白WB和磷酸化蛋白体外降解问题

1.最近想做水孔蛋白磷酸化影响植物吸水实验，但是原生质体按照文献中cut into small pieces that were finally transferred to 3 ml enzymatic digestion medium (ES-500M: 500mM mannitol, 10 mM MES/KOH (pH 5.8), 10 mM CaCl2, 10 mM KCl) plus 1% (w/v) cellulase Onozuka R10 (Duchefa, Haarlem, The Netherlands) and 1% (w/v) macerozyme R10 (Duchefa, The Netherlands The protoplasts were prepared in the dark with gentle shaking for two to three hours, filtered through a 50-m nylon filter, centrifuged at 80 g at 4 °C for 5 min, and washed three times with ice-cold ES-500M without enzymes. Protoplasts were stored in the dark on ice for at least 30 min before an experiment was started. 酶解3小时并没有原生质体出来。不知道是什么原因？

由于磷酸化蛋白容易降解，不知道酶解时间长了会不会影响磷酸化蛋白的降解，从而影响结果。想问一下离体植株磷酸化蛋白体外多长时间降解呢？

2.做磷酸化蛋白的WB 提取蛋白后可以在-20 保存多长时间？还是必须提取完之后立刻做呢？