《常用荧光探针小结课件》由会员分享，可在线阅读，更多相关《常用荧光探针小结课件（25页珍藏版）》请在金锄头文库上搜索。
1、1,常用荧光探针小结 一、异硫氰酸荧光素（Fluorescein isothiocyanate, FITC） FITC有两种异构体，性质稳定，低温下干燥保存，其性状多年不变，室温下也能保存两年以上。异构体I、II均能与蛋白质良好结合，但异构体I的荧光效率更高，与蛋白质的结合也更稳定。 FITC的最大吸收光谱为490-495纳米，最大发射光谱为520-530nm，呈明亮的黄绿色荧光。FITC含有异硫氰基，在碱性条件下能与IgG的自由氨基（主要是赖氨酸的-氨基）形成荧光抗体结合物。,2,Exitation max: 495 nm; Emission max: 519 nm; Solvent pH:
2、8.00,3,Immunocytochemistry/Immunofluorescence-alpha Tubulin antibody DM1A (FITC) (ab64503) ICC/IF image of ab64503 stained human HeLa cells. The cells were methanol fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab64503, 1g/ml, FITC conjugated (green) for 1h
3、 at room temperature. 1% BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. Alexa Fluor 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).,4,二、荧光素酯,Carboxyfluorescein diacetate succinimidyl ester (CFDA
4、-SE) is a cell permeable dye generally used in animal cell proliferation research. CFDA-SE enters cells by diffusion and is cleaved by intracellular esterase enzymes to form an amine-reactive product, CFSE. This product produces a detectable fluorescence and covalently binds to intracellular lysine
5、residues and other amine sources.,酯酶,5,视频：The Use of Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) to Monitor Lymphocyte Proliferation,6,四甲基异硫氰酸罗达明，它是一种紫红色粉末，较稳定。其最大吸收光谱为550nm，最大发射光谱620nm，呈橙红色荧光，与FITC的黄绿色荧光对比清晰，与蛋白质结合方式同TITC。它可用于双标记示踪研究。,三、异硫氰酸罗丹明（TMRITC）,7,Detection of -tubulin in A549 cel
6、ls demonstrates use of rhodamine-labeled secondary antibody. Cells were probed with a mouse anti-tubulin primary antibody (0.4g/mL) and Rhodamine-goat anti-mouse secondary antibody (2g/mL). Nuclei were labeled with Hoechst Dye. Images were acquired by fluorescence microscopy. A. Fluorescence image s
7、hows a delicate network of -tubulin (pseudo-colored green) located exclusively in the cytoplasm. B. Nuclear counterstain with Hoechst Dye (pseudo-colored blue) C. Merged image.,8,AC41323-0010,RB200，也称丽丝胺罗丹明B 无定形褐红色粉末，不溶于水，易溶于酒精和丙酮，性质稳定，可长期保存，最大吸收光谱为570nm，呈明亮的橙色荧光，因与FITC的黄绿色有明显区别，故被广泛用于对比染色或用于两种不同颜色的
8、荧光抗体的双重染色。 标记方法方法：取1g RB200及五氯化磷（PCL5）2g放乳钵中研磨5min（在毒气操作橱中），加10ml无水丙酮，放置5min，随时搅拌，过滤，用所得溶液进行结合。将每亳升血清用1ml生理盐水及1ml碳酸盐缓冲液（0.5mol/L，pH9.5）稀释，逐滴加入0.1ml RB200溶液，随加随搅拌，在0-4继续搅拌 12-18h。,四、罗丹明200,9,Confocal image of double immunostaining for Akt in hippocampal CA1 pyramidal neurons. Section is shown from a
9、normal rat. The red color derived from lissamine rhodamine conjugated secondary antibody represents MAP2, the green color derived from fluorescein indicates the labeling of Akt. Yellow represents overlay of red and green.,10,五、溴化乙锭 详见第四节“应用于核酸检测的荧光探针技术”,11,六、DAPI （ 4,6-diamidino-2-phenylindole）,DAPI
10、 was first synthesised in as part of a search for drugs to treat trypanosomiasis. Although it was unsuccessful as a drug, further investigation indicated it bound strongly to DNA and became more fluorescent when bound. When bound to double-stranded DNA DAPI has an absorption maximum at a wavelength
11、of 358nm (ultraviolet) and its emission maximum is at 461nm (blue). Therefore for fluorescence microscopy DAPI is excited with ultraviolet light and is detected through a blue/cyan filter. The emission peak is fairly broad DAPI will also bind to RNA, though it is not as strongly fluorescent. Its emi
12、ssion shifts to around 500nm when bound to RNA.,12,DAPI (magenta) bound to the minor groove of DNA (green and blue).,DAPI can be used for fixed cell staining, the concentration of DAPI needed for live cell staining is generally very high and rarely used for live cells. Though it was not shown to hav
13、e mutagenicity to E. coli, it is labelled as a known mutagen in manufacturer information. As it is a DNA binding compound it is likely to have some low level mutagenic properties and care should be taken in its handling and disposal.,13,A type of simple fluorescence: DAPI is excited in near UV with
14、365 nm and emitted in the violet-blue spectral range. The marking reveals cell nuclei and particularly the chromosomes,DAPI is a dye which binds to DNA and shows an increased blue fluorescence when bound to DNA. The blue fluorescent spots in the picture show the nuclei in the mycel.,14,Endothelial c
15、ells under the microscope. Nuclei are stained blue with DAPI, microtubles are marked green by an antibody and actin filaments are labelled red with phalloidin.,15,七、Hoechst,Hoechst stains are part of a family of blue fluorescent dyes used to stain DNA. These Bis-benzimides were originally developed
16、by Hoechst AG. There are three related Hoechst stains: Hoechst 33258, Hoechst 33342, and Hoechst 34580. The dyes Hoechst 33258 and Hoechst 33342 are the ones most commonly used and they have similar excitation/emission spectra.,16,17,Transmission image of HeLa cells, with overlay of Hoechst 33258 staining (blue).,Hoechst 33258 (magenta) bound to the minor groove of DNA (green and blue),18,八、碘化丙啶(Propidium Iodide, PI）,Propidium iodide is an intercalating agent and a fluorescent molecule t
