在细胞里 tdTomato有严重的aggregation,不过在细胞质里也还是有弥散状分布的tdTomato.但是似乎bleaching 和quenching只发生在弥散分布的tdTomato蛋白里,aggregation的亮点似乎没有太大的荧光衰减.难道aggregation有助于蛋白稳定?更稳定的结构保证荧光集团的稳定? 在ZIESS网页: zeiss-campus.magnet.fsu.edu/articles/probes/anthozoafps.html (page 3/18)中提到 最初的DsRed是四聚体,荧光较稳定,但是不适合实际应用.在实验室产生单体RFP(red fluorescence protein)非常难,使用了不断突变.第一代单体RFP荧光较弱,而且非常容易photobleaching.RFP和DsRed主要区别就是一个是单体一个是四聚体.看来寡聚化(oligomerization)对荧光稳定性是有帮助的. -------------------------------------------------------------------------- (1)pH 今天我测了所有接触样品的试剂的pH.固定液多聚甲醛PA,和Carboiimide(EDC),以及清洗试剂PB都是pH7.4,在这个pH mCherry应该是稳定的.所以我想pH的因素可以排除. (2)光强 试试减弱激发光的光强. 按照今天的实验结果来看,光强是目前影响最大的因素. 以前小水獭想尽量减少曝光时间,总觉得曝光时间越长,荧光衰减越快. 但是减弱光强,单位时间内荧光集团接受能量减少,从今天的结果来看,是有利于荧光的稳定的. (3)抗氧化剂 谢谢徐磊的指点,提醒我还有一条路就是加抗氧化剂. 在Tsien R 1998 的review里也提到了Trolox, 维他命E衍生物,可用于荧光保护. Cell-permeant antioxidants may be helpful in protecting such GFPs from bleaching. An example is Trolox, 6-hydroxy-2,5,7,8-tetramethylchroman-2- carboxylic acid, a water-soluble vitamin E analog commercially available from Aldrich Chemical Co., Milwaukee, WI. 我搜到了另一篇文章 Campos LA, Liu J, Wang X, Ramanathan R, English DS, Munoz V (2011) A photoprotection strategy for microsecond-resolution single-molecule fluorescence spectroscopy. Nature methods 8:143-146 是使用Trolox的. (4)封片试剂 mounting medium Tsien 1998 里提到了FPs对酸和有机试剂敏感,我就开始紧张封片试剂mounting medium了. 刚好搜到下文: Malkani N, Schmid JA (2011) Some secrets of fluorescent proteins: distinct bleaching in various mounting fluids and photoactivation of cyan fluorescent proteins at YFP-excitation. PLoS One 6:e18586. 文章用CFP和YFP测试两种封片试剂来自于Dako和ultramount,不过我们实验室用的是Vector公司的mountingshield.文中提到,目的是保护荧光的封片试剂,其中的有机溶剂,主要是甘油glycerol可能会影响FP. 文中还提到最佳的保护溶剂是PB:glycerol=1:7.小水獭腹黑的笑了,以为找到了终极秘籍. 今天小水獭对照试验了三种封片试剂(1)PB,(2)mounting medium (3) PB+glycerol,坑爹啊!!!!!!!!!!!!! PB最好,mounting medium 其次,他们建议的PB+glycerol最不灵,片子毁得一塌糊涂,背景高的吓人.信号却没了 不过目前小水獭认为,对fixed tissue,封片试剂确实不能提高荧光稳定性,哦..........还不如PB. 但是对于化学合成的抗体偶联荧光分子,比如Alexa,Texasred 系列,封片试剂应该是有帮助的. (5)新鲜配置4%PA Dr.Pan建议以后最好每周新配PA,小水獭想也许应该这样的.
This years Nobel Prize of Chemistry went to three scientists who made significant contribution toward marking proteins in the living cell with Green Fluorescent Protein (GFP). Nobel prizes usually cause a sensation in the media but not among real researchers. Since if the discovery could be bestowed with the honor, it has already been widely known or widely used for quite a while. Now what researchers really care is what will come after Green Fluorescent Protein technology since GFP certainly has some limits. There are still many urgent demands from biologists which GFP can not meet. Current technology of protein dyes The revolution of GFP is to enable researchers to see when and where certain proteins are expressed. You could even associate the intensity of GFP with the protein concentration. Then you can derive some quantitative measurements for your modeling. The most impressed GFP image which I have seen is from Robert Kays research of the Chemotaxis of E Coli. He showed that when certain genes of molecular motors are knocked out, the cell can not move at all even GFP images show signaling proteins are correctly synthesized in the chemical gradient direction. Fig. 1 GFP to show the expression of Chemotaxis signaling proteins You can see a video here: http://www2.mrc-lmb.cam.ac.uk/groups/rrk/movie2.html Gene Myers from Janelia Farm Research campus of HMMI even introduced computerized visual identification to scan thousands of GFP images from genome-wide experiments. Therefore, not just single biochemical pathway could be made clear from GFP. The total protein interaction network dynamics could be inferred from genome-wide protein expression pattern with GFP. Limitation of protein dyes Protein dyes technology is hugely improved fby the mutations leading to various colors and increased Fluoresce in the Roger Tiens hands. However, the number of different color is still too limited with the respect to the increasing demands from biologists to see the expression patterns of the large number of different proteins simultaneously. We could learn when and where proteins are expressed by GFP but the number is usually very small. Fig. 2 Three color scheme GFP image for cell mitosis To make up for this limitation, the current high-throughput technology is usually used to quench cells and extract cell extracts every few minutes or even hours to capture the cell dynamics. Then concentrations of different proteins are separated by mass-spectrometer. However, there are several disadvantages: First, we lose the information of spatial distribution of those proteins. Secondly, it is highly efforts-consuming job so it is quite coarse-grained in time resolution. Thirdly, there certainly are some unexpected changes when we crash the cells. Therefore, high-throughput mass-spectrometry technology can handle with hundreds of proteins at the same time but lose much information of space and time. Outlook of protein marking technology It is urgent to develop a new technology to mark hundreds of different proteins simultaneously and to detect their expression without any damage to the cell. There are many wild speculations. Nobody has really developed some feasible technology, yet. One possibility is to use some proteins with hundreds of different conformations if it is mutated. They can be attached to any proteins which the biologists are interested. Then an nano-NMR array scanner could be used to scan the cell for its spatial-temporary protein expression pattern. There is no need for the nano-scanner to have a high resolution. It is enough if it can distinguish various shapes of mutated protein markers. However, it has to be fast enough to give a better time resolution. Feynman once said that what Biology needs is to see better at the atomic Level: We have friends in other fields--in biology, for instance. We physicists often look at them and say, You know the reason you fellows are making so little progress? (Actually I don't know any field where they are making more rapid progress than they are in biology today.) You should use more mathematics, like we do. They could answer us--but they're so polite, so I'll answer for them: What you should do in order for us to make more rapid progress is to make the electron microscope 100 times better. This observation is still valid if we want to study the details of the biochemical reactions. However, if we want to study the dynamics of hundreds or even thousands of proteins within one cell, a automatic protein marking and detecting technique with high spatial-temporary resolution is what biologists desperately need. This years Nobel Prize does not signify the beginning of the end of protein marking technology; instead, it is just the end of the beginning. END
标签: GFP
