FITC标记蛋白质.doc

FITC标记蛋白质 Product Numbers: Synonym: FITC Appearance: yellow powder Storage Temperature 2-8 °C Molecular Formula: C21H11NO5S Molecular Weight: 389.4 Excitation: λmax = 495 nm Emission: λmax = 525 nm Fluorescein isothiocyanate (FITC，异硫氰酸荧光素) is widely used to attach a fluorescent label to proteins via the amine group. The isothiocyanate group reacts with amino terminal and primary amines in proteins. It has been used for the labeling of proteins including antibodies and lectins. FITC-N=C=S? +? N-H2-protein??→? FITC-NH-S-C-N-H-protein Preparation Instructions FITC is tested for solubility and solution appearance at 1 mg/mL in acetone to give a clear yellow solution. It is soluble in anhydrous dimethyl sulfoxide (DMSO) at 5 mg/mL. It is soluble in water at less than 0.1 mg/mL in water, at 20 mg/mL in ethanol and at 9 mg/mL in 2-methoxyethanol. An organic solvent for stock solution is advised, since FITC decomposes in water. FITC is diluted in basic buffer for coupling procedures immediately prior to use. Storage/Stability The products are light-sensitive, and should be stored dry and in the dark at 2 °C to 8 °C. Procedure of Labeling Protein with FITC Preparation before experiment: 配100mL，0.1M PH=8.5的SB缓冲液(0-5℃保存，且不超过一周，最好现配现用)； 准备好A,B,C,D四个石英比色皿； 取一小容量瓶，事先用锡箔纸将其完全包住以避光； 用超纯水注满D=15mm，L=300mm的柱层析分离吸附柱，过夜浸泡，同时检测其是否漏液，第二天再用PBS浸泡7-8h； 用DIW浸泡葡聚糖Sephadex G-50过夜(1g Sephadex G-50配5mLDIW)，使其溶胀，打开孔状结构； 而后将DIW倒掉，注入适量PBS(PH=7.4)，用锡箔纸盖好防灰，4℃保存；注：(1) Sephadex G-50量可以过多，过完柱层析分离吸附柱后剩余部分可加入 0.02%(m/v)NaN3，防止细菌，保存于4℃冰箱中； (2) Sephadex G-50第一次配好后可以反复使用多次，但应做相应后处理(见后)； (3) Sephadex G-50分离范围：1000-30,000(分子量)， 计算Protein中游离-NH2，配制1.0mg/mL Protein/SB蛋白溶液：BSA, Fraktion V Mw=67000Da, Arg 26个; lys 60个; 20mg BSA; 2mLSB(0.1M PH=8.5)；游离-NH2：n1=20×0.001g×(26+60)/( 67000g.mol-1) =2.57×10-5mol；溶于小容量瓶中(外包锡箔纸避光处理)，加搅拌子搅拌，使其完全溶解； 注：组成蛋白质的20中氨基酸中，仅Arg，lys在侧链上有游离的-NH2。 3. 求算FITC的量： 5.8mg FITC，5.8mLDMSO(干燥)，溶于5mL冻溶管中； 注：(1)