Go to Hampton Research website to check materials for protein crystallization.
Normally, you need to get pure protein first;
then do crystal screening to see if you could get protein crystal;
then optimize the crystallized conditions to get good single crystal;
then collect X-Ray diffraction data at synchrotron beamline at Beijing or other synchrotron facilities all over the world, or lab-based X-ray equipment;
then solve the protein structure by softwares(if you really want to do the thing, be familiar with softwares, such as CCP4, CNS, SHELX, COOT, solve/resolve and others, from now on. Some softwares are free to academia so you can download for their websites, but some not free, if you boss has enough money, just ask to buy, but I think many bosses in China are miser).
then deposit you structure to PDB (protein data bank), and publish your paper if you get the structure. If you can do the relative functions, that is better. Structure plus function is the best. A very good paper will you get.
Anyway, the experiments are routine. You have to spend more time on softwares if you want to be an expert.
Besides, I think you need to read some textbooks about protein crystallography. Basic knowledge is necessary.
Many companies have commercialized screening kits, such as Hampton Research, Emeralds, Qiagen. If you do not want to buy the Hampton Research screening kits, you could prepare the kits according to the formula. But the total cost for buying reagents would be more than that of buying the screening kit. The best way is to co-operate with the labs which do protein crystallography because of your lab's condition. Some labs in Beida, Qinghua, and Institue of Biophysics do that job.
If you have other questions later, just free to ask.
What tag did you use for your protein? Histag, GST, MBP or others? You have to check the purity with SDS-PAGE after every purification step, such as Ni-NTA column, Ion-exchange, and size column. As xsvulture said, the purer, the better.
There is one thing you may be confused. The 95% purity on SDS-PAGE does not just mean enough purity. Because the purity on SDS-PAGE is based on denatured form. The 95% purity should include the homogeneity of three-dimensional shape and the charge distribution of protein.
After you get good purity (>95%) on SDS-PAGE, you maybe need to run a size-column to check the apparent molecular weight of your protein to see if it is dimer, tetra-mer or aggregate(for example, big M.W. more than one million) (In aggregate case, you won't get any crystal even though you can get only one band on SDS-PAGE. I met the case before). This way is crude.
There are other ways to check the homogeneity of protein in certain buffers, such as Dynamic Light Scattering and thermofluor method. You can check the references later if you really need to do these experiments.
Just do crystallization as the references' method first. If no crystal, do crystal screening.
How much is the sequence identity of your RNaseA to the crystallized one? I think that there should be structure of the latter in PDB and maybe you could use Molecular Replacement to solve the structure of your RNaseA if you could get crystal later.
最新回复
bamboo16 (2013-7-31 17:57:39)
建议联系清华大学的施一公老师。
abc816 (2013-7-31 17:58:00)
虽然结晶是个粗活，不过粗中有细，自己看文献是不行的，隔行如隔山，必须和结构生物学实验室合作。
清华、北大、生物物理所、科大、生化所都有。
standbyme (2013-7-31 17:58:18)
PINK (2013-7-31 17:58:42)
Go to Hampton Research website to check materials for protein crystallization.
Normally, you need to get pure protein first;
then do crystal screening to see if you could get protein crystal;
then optimize the crystallized conditions to get good single crystal;
then collect X-Ray diffraction data at synchrotron beamline at Beijing or other synchrotron facilities all over the world, or lab-based X-ray equipment;
then solve the protein structure by softwares(if you really want to do the thing, be familiar with softwares, such as CCP4, CNS, SHELX, COOT, solve/resolve and others, from now on. Some softwares are free to academia so you can download for their websites, but some not free, if you boss has enough money, just ask to buy, but I think many bosses in China are miser).
then deposit you structure to PDB (protein data bank), and publish your paper if you get the structure. If you can do the relative functions, that is better. Structure plus function is the best. A very good paper will you get.
Anyway, the experiments are routine. You have to spend more time on softwares if you want to be an expert.
Besides, I think you need to read some textbooks about protein crystallography. Basic knowledge is necessary.
cocacola (2013-7-31 17:58:59)
楼上说的不错，
首先难道足够纯的蛋白，越纯也好结晶，最好能95%以上。
如果有钱，买Hampton等公司的筛选试剂盒，不是一般的贵，如果你只是做一两个蛋白，估计谁都会心痛的。
不然就参考文献，自己配些buffer，根据文献，对pH，PEG，盐浓度等一些了条件做调整，筛选。
养晶体之后就是看晶体，不要把盐晶当成是蛋白晶体。
如果有蛋白晶体，捞出来去做x-ray。
数据分析可能是最难的一步了，反正我是不懂，我们公司有专门的人做这一块的，我看着是云里雾里，哈哈。
祝好运！
PINK (2013-7-31 17:59:16)
Many companies have commercialized screening kits, such as Hampton Research, Emeralds, Qiagen. If you do not want to buy the Hampton Research screening kits, you could prepare the kits according to the formula. But the total cost for buying reagents would be more than that of buying the screening kit. The best way is to co-operate with the labs which do protein crystallography because of your lab's condition. Some labs in Beida, Qinghua, and Institue of Biophysics do that job.
If you have other questions later, just free to ask.
jiushikeshui371 (2013-7-31 17:59:35)
请高手们指教
PINK (2013-7-31 17:59:53)
What tag did you use for your protein? Histag, GST, MBP or others? You have to check the purity with SDS-PAGE after every purification step, such as Ni-NTA column, Ion-exchange, and size column. As xsvulture said, the purer, the better.
There is one thing you may be confused. The 95% purity on SDS-PAGE does not just mean enough purity. Because the purity on SDS-PAGE is based on denatured form. The 95% purity should include the homogeneity of three-dimensional shape and the charge distribution of protein.
After you get good purity (>95%) on SDS-PAGE, you maybe need to run a size-column to check the apparent molecular weight of your protein to see if it is dimer, tetra-mer or aggregate(for example, big M.W. more than one million) (In aggregate case, you won't get any crystal even though you can get only one band on SDS-PAGE. I met the case before). This way is crude.
There are other ways to check the homogeneity of protein in certain buffers, such as Dynamic Light Scattering and thermofluor method. You can check the references later if you really need to do these experiments.
Just do crystallization as the references' method first. If no crystal, do crystal screening.
How much is the sequence identity of your RNaseA to the crystallized one? I think that there should be structure of the latter in PDB and maybe you could use Molecular Replacement to solve the structure of your RNaseA if you could get crystal later.
Good luck!
PINK (2013-7-31 18:00:14)
電磁波理論與晶體學都應該看看.
蛋白質晶體結構方面的有一本簡易的﹐<<晶體﹐X射線和蛋白質>>﹐書有點老﹐可以
用來上手。 此書在從電磁波到晶體X-ray衍射的數學推導方面有一定的學習價值﹐而晶體
方面的內容過于簡略。新手應該合適的。
也可以從網上找protein crystallography的教材來看﹐英文的很多。
caihong (2013-7-31 18:00:40)
请高手们指教
===========================================================================================================
好像不是那么回事吧。国外一个教授跟我说，就是再同源的蛋白，你也不大可能用结晶一个蛋白的条件去拿到另一个蛋白的晶体。
还有，你要专注于某一个蛋白去做的话，会比较困难吧，从表达，到纯化，到结晶，到优化，中间任何步骤都是淘汰率很高的。所以建议跟你老板商量多做几个蛋白，不要死盯一个蛋白。
我也是刚刚开始做，之前到别的实验室做，已经试过几个蛋白了，可惜只拿到了盐晶体，当时还以为是蛋白呢，结果空欢喜了。现在回到自己实验室，定购了一些试剂耗材的，又打算弄些蛋白做，感觉风险蛮大阿。。。
PINK (2013-7-31 18:01:04)
~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
道理是如此﹐但也可以一試的﹐蛋白結晶無定則。
PINK (2013-7-31 18:01:31)
~~~~~~~~~~~~~~~~~~~~~~~
蛋白溶液用什麼buffer? 磷酸鹽?
jiushikeshui371 (2013-7-31 18:01:48)
PINK (2013-7-31 18:02:08)
1. 你是指方法上可行與否﹐還是指真核生物蛋白在原核生物中表達後會其結構是否
發生改變而失真?
方法上是可行的。這有個前提﹐蛋白是可以自我自動折疊的(但有時也要伴侶
的協助)。我現在做的蛋白都是人類蛋白﹐基本都是用Ecoli表達折疊成功的。MBP對
蛋白的折疊效果比GST好。但是﹐如果蛋白涉及表達後修飾話﹐Ecoli不一定合適﹐
你可以用Yeast(我沒做過) 或者insect cell(我做得不多)來表達﹐而insect cell
的表達量比不上Ecoli的﹐而且所用營養基貴。一般來說﹐若果在Ecoli中只能得到
包涵體﹐沒辦法用tag來解決水溶性的問題﹐我才會考慮用insect cell。 如果表達
的是膜蛋白﹐在純化時﹐可利用表面活性劑來提取。
你可能擔心結構會改變。蛋白是自動折疊的﹐而且它的結構已經由其序列決定
了(現在就有人在做從蛋白序列預測其三維結構的理論計算研究)﹐在原核中表達得
到的結構跟在真核中得到的不會有什麼差別﹐關鍵是可否折疊可溶。
2. 你可以留histag 在上面﹐也可切除。有時候﹐histag並不影響結晶。我記得我
第一個蛋白晶體就是沒有切掉histag的﹐在N- 端﹐而且有20個多餘的雜氨基酸。也
有人做過﹐留histag做晶體篩選時﹐沒有結晶﹐把histag去掉後﹐能得到晶體。一
般來說﹐histag對X-ray衍射沒有影響﹐得到的結構是看不到histag的﹐在晶體中﹐
每個蛋白單體上的histag的取向是雜亂的。 histag 對結晶的影響是沒有定論的。
在做蛋白結晶時﹐不要預設定論﹐除了些大概流程原則。
如果你想將蛋白弄乾淨點﹐也可每次都切掉histag。你要用thrombin?還是TEV?
xue258 (2013-7-31 18:02:26)
popo520 (2013-7-31 18:03:02)
~~~~~~~~~~~~~~
蛋白溶液用什麼buffer? 磷酸鹽?
恩，结晶试剂里边有磷酸铵，然后我的那个蛋白是一种需要镁离子参与的酶，所以我加入了镁离子，期望可以辅助结晶。最后发现不加镁离子就得不到晶体，而只加镁离子不加蛋白却可以得到晶体。所以结论就是磷酸镁盐晶。
66小飞侠 (2013-7-31 18:04:13)
66小飞侠 (2013-7-31 18:04:37)
我觉得盐晶长得还是和蛋白晶体不太一样的，面少，比较薄一点。
恩，我上次也是，优化没两天长了很漂亮的晶体，当时也把我高兴坏了。哎可是后来大牛一看说可能是盐晶，后来用甲基兰染色也没有着色，才确定是盐，可怜啊，空欢喜一场，希望以后大家一起多交流阿！
jiushikeshui371 (2013-7-31 18:05:02)
还有两个问题要请教：
第一个是我现在是用His柱子纯化蛋白（GE公司的预装柱），但纯化后杂蛋白还有，量不大，但达不到结晶的要求（文献上说结晶时电泳纯，即电泳时仅一条带），请问，蛋白纯度必须这么纯吗？如果那么纯的话，过完His柱子后是否还要经过其它的纯化方式？
第二个问题是：正如我刚才所说，用His纯化蛋白，然后用含有咪唑的Elution buffer洗脱目的蛋白，这样蛋白液中就含有咪唑了。做蛋白结晶时是否必须把咪唑去除，另外，结晶时的缓冲液是用什么？
多谢您了。
66小飞侠 (2013-7-31 18:05:33)
QUOTE:
呵呵，我们这边实验室一般都再过一个离子交换柱和分子筛柱，即用FPLC进一步纯化的，过完分子筛，咪唑也就去掉了
【求助】蛋白结晶