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生物学实验都是些看不见,摸不着的东西,所以基本上都是使用这个方法,看不见的,我们拿看的见的去标记它,测不着的或者不好测的,拿可以测容易测的去标记它。很多的实验手段,其实最初都是很简单的,为了提高灵敏度和精度,不断改进,越来越烦琐,抑或者出现了一些分支。通常这时候,最初那个简单的,最根本的东西就被人所忘记。看到的尽是表面上的细节。至少现在的书太二了,本来基本上一样的实验,阐述原理的时候被描成了两样的东西。

同位素标记,荧光标记自不用说。芯片实验也是标记,array上的spot序列是已知的,它会跟什么基因杂交,也是已知的。我们并不知道会有什么基因表达,但是当抽提的东西,跟某个spot有杂交信号的时候,我们就知道某个基因有表达。这就是标记。还有我们需要知道表达的量,这个依然不好测。同样也是使用标记,用cy3或cy5来标记,通过检测荧光的强度,就知道了表达的量。

所有的杂交实验都是标记,southern blot, northern blot, western blot都是。不管是核酸还是蛋白,不管它是用什么介质。都是用一个已知的,去标记一个未知的。通过测已知来估计未知的东西。

跑胶的时候,拿去紫外灯光下看。同样也是因为标记。DNA我们是看不到的,但是在DNA下嵌入了EB,可以在紫外灯光下看到EB,所以我们也就看到DNA。

所有的切片,都是在做标记,不管是用于光镜还是电镜,其实电镜本身和光镜是没本质区别的,因为可见光的波长范围很有限,电子也是一种波,成像原理是一样的,只是我们不能直接看到而已。使用电子是为了突破可见光的局限而已,使用光镜需要对细胞进行染色,使用电镜也是一样,不过染的不是染料,而是金属。

测蛋白,用考马斯亮蓝染。在280nm有吸收,那是因为苯环的共轭电子对。虽然不是标记上去的,但也可以这么想,反正是测一个我们已知的东西,来估计未知道的东西。

DNA测序,给四种碱基标上不同的荧光。

还记得脂肪代谢是怎么被揭开的吗?使用接了苯环的脂肪酸喂马,收集马尿去检验,因为马不能代谢苯环。所以拿苯环去标记脂肪酸。

还有那个DNA还是蛋白是遗传物质的实验,肺炎双球菌实验。分别用S和P的同位素标记蛋白和DNA。

还有一些看似不是标记的,其实也是标记,比如酵母双杂交,因为转录因子都有一个DNA-binding domain和一个RNA-binding domain,我们并不能直接检测到蛋白X和Y是否有相互作用,但我们可以表达溶合蛋白DBD-X和RBD-Y,如果XY有相互用用的话DBD和RBD就会在一起,就是一个有功能的转录因子,就可以转录报告基因,检测到报告基因,就表明了XY有相互作用,这就是标记,拿DBD和RBD去标记X和Y,通过DBD和RBD的行为去估计X和Y的行为。

DNA重组也使用标记,我们无法直接检测到是否重组了,是否在需要的位点重组了,所以使用了报告基因来做标记,检测到报告基因了,表明重组了,使用抗药基因,在加了药物的培养基上筛选,活下来的表明重组正确。当然有假阳性。抗药基因也是“标记"。

基本上绝大多数的实验原理,最根本的一个想法,就是使用一个已知的,或者容易检测的,去标记一个未知的,或是难以检测的。

实验的手段太多了,很多人搞不清原理。做实验的人,通常就是照着实验手册,一步一步在加东西而已。只要稍微思考一个,很多东西,其实不用记。细节的东西不需要记,需要的时候查就行了,当是基本的原理还是需要理解的。不然白学了。

很多人觉得生化就是一堆描述性的实验结果。其实很多东西也是不需要记的。举个例子,比如说DNA转录的时候,组蛋白会被乙酰化,这个细节很多人就是死记下来的,当然国内的书太二了,很多书没说乙酰化的位点是Arg和Lys,不过告诉这个事实,很多人还是死记下来。其实想一下,理解了,就不用记。为什么是这两个AA,而不是其它的?组蛋白之所以带正电就是因为富含这两个AA,这两个AA都有-NH,带正电,正是由于乙酰化了这两个位点,屏蔽了正电,DNA是带负电的,所以乙酰化之后正负电引力变小了,结构就松散了。有利于转录。这个东西就变得理所当然一样,根本不需要记。 

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