结果如上图所示:
作者推理RY1/RY2两段短的核酸序列(motif)是FUS3蛋白的结合作用位点,为了证明作者的观点,作者采用凝胶阻滞实验加以论证( electrophoretic mobility shift assay),用含有RY motif并且进过放射性标记的探针同FUS3蛋白孵育后电泳,下面是图下的注解,我有些看不太明白,还请高人指点---
图中原文注解如下:Section B shows an electrophoretic mobility shift assay experiment with RY1 site. A radiolabeled probe carrying the RY1 site (TGCATGCATG) was incubated with the FUS3 protein present in bacterial extracts. Lane 1, radiolabeled probe only; lane 2, rabiolabeled probe incubated with FUS3 extracts; lanes 3
and 4, radiolabeled probe incubated with FUS3 extracts after incubation with an excess of 100- and 500-fold, respectively, of unlabeled probe; lanes 5 and 6, same as in lanes 3 and 4 except that the unlabeled probe carried a mutated RY1 site (mRY1). Section C is similar to section B except that the radiolabeled probe (lane 1) carried the RY2 site (CATGCATG). The probe is retarded by FUS3 extracts (lane 2) and competition experiments were performed with an unlabeled probe carrying the native
(lanes 3 and 4) or mutated (lanes 5 and 6) RY2 site (mRY2). The arrows indicate FUS3-RY complexes.
有几点不明白:
1,泳道3, 4的描述究竟是什么意思,是分别是用100和500倍足量的探针孵育;还是分别用未经标记的探针孵育;还是一个用足量探针孵育(为何一个泳道用两个数据),一个用未经标记的探针孵育;
2, 5,6泳道是RY1序列突变后与FUS3蛋白孵育的,按理说突变后不会结合才是,但图片显示是结合上去了,箭头指明出现杂交带。
3,我个人理解,没有放射性标记的探针在同样条件下,应该不会显示出来条带,为什么实验中用这种探针都有条带呢?
4,这种实验需要一般需要设置怎样的对照才合理,大体上我缺乏把握。
俺英语差劲,还请各位同辈前辈指导指导---