FITC标记蛋白

FITC标记蛋白 FITC标记蛋白质 Product Numbers: Synonym: FITC Appearance: yellow powder Storage Temperature 2-8 ?C Molecular Formula: CHNOS Molecular Weight: 389.4 21115 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-H-protein ? FITC-NH-S-C-N-H-protein 2 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 1. Preparation before experiment: 1) 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); 2) 准备好A,B,C,D四个石英比色皿; 3) 取一小容量瓶，事先用锡箔纸将其完全包住以避光; 4) 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; 5) 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)， 2. 计算Protein中游离-NH，配制1.0mg/mL Protein/SB蛋白溶液: 2 BSA, Fraktion V Mw=67000Da, Arg 26个; lys 60个; 20mg BSA; 2mLSB(0.1M PH=8.5); -1-5游离-NH:n=20×0.001g×(26+60)/( 67000g.mol) =2.57×10mol; 21 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-NH。 2 3. 求算FITC的量: 5.8mg FITC，5.8mLDMSO(干燥)，溶于5mL冻溶管中; 注:(1) 配好后分装于0.5mL离心管中，-69?C保存，之后可以直接使用。 (2) FITC的量应适中，以使蛋白质中所有游离的-NH均与FITC反应， 2 FITC量较少 ? 未反应-NH较多 ? F/P低; 2 FITC量过多 ? F/P高; (2) 对BSA，取已配好的150uL 1.0mg/mL FITC/DMSO， -6故FITC:n=0.15×1.0×0.001/389.4 mol =0.385×10mol 2 Dissolve the FITC in anhydrous DMSO at 1.0mg/mL. Note: This should be prepared fresh for each labeling reaction. 4. FITC标记Protein反应: 用移液器每次取10ul，间隔、缓慢加入BSA/SB蛋白溶液中; 全部加完后，搅拌、混合一会，转入4?，黑暗中反应8h(冰箱内); 注:加入FITC的过程中应注意避光。 For each 1 mL of protein solution, add 50 mL of FITC solution, very slowly in 5 mL aliquots while gently and continuously stirring the protein solution. After all the required amount of FITC solution has been added, incubate the reaction in the dark for 8 hours at 4 ?C. %0.15. 测1.0mg/mL Protein/SB蛋白溶液在280nm处的吸光系数E。 配制3mL 1.0mg/mL BSA/SB蛋白溶液(5mL冻溶管中)，用“UV-7504紫外可见分光光度计” %0.1测其在280nm处的吸光系数E。 A:3mL SB，按“0ABS/100%T”清零(清零时用相应蛋白质溶液的溶剂); %0.1B:3mL 1.0mg/mL BSA/SB蛋白溶液，测其在280nm处的吸光系数E; 后处理:A:倒掉3mL SB，DIW冲洗，烘干; B:将BSA/SB蛋白溶液取出，放回原来5mL冻溶管中，4?C保存， 用SDS浸泡石英比色皿B，而后用DIW冲洗，烘干; 6. 加NHCl终止反应 4 Add NHCl to a final concentration of 50 mM, mix for a while then incubate for 2 hours at 4 ?C.4 -3NHCl:m=50×0.001mol/L×(2.0+0.15)×0.001L×53.49g/mol=5.75×10g=0.00575g;41 7. Add xylene cyanol to a final concentration of 0.1%(m/v) and glycerol to 5%(m/v). xylene cyanol 0.0042g，显色作用，使过柱时蛋白质与FITC易于分辨; (本实验未加，最下层为蛋白质) glycerol 甘油 m= (2.0+0.15) mL×5%=0.1075g (2mL离心管称取) 2 搅拌，混合5min左右; 8. 装填柱层析分离吸附柱 1) 取45mL冻存管，用锡箔纸包好以避光; 2) 湿法装柱:(装填过程中确保PBS液面比Sephadex G-50高，以防混入空气而产生气泡) 先在柱子内注入3/4左右的PBS，而后加入Sephadex G-50/PBS溶液;分批次、缓慢加入柱子内，最后用塑料滴管吸取，沿着柱子四壁旋转，逐步、缓慢地加入，装填至近满，留下4-5cm的空间以待注入BSA-FITC荧光蛋白溶液; 3) 在装填的整个过程中打开活塞(淋出液用大烧杯承接)，同时用玻璃棒+橡胶塞从下到上轻轻敲打柱子，使Sephadex G-50装填紧密，注意使Sephadex G-50柱子内不得有气泡，不得有断层，使柱子竖直，确保顶部Sephadex G-50端面水平; (故滴加Sephadex G-50时应旋转加入，不得直接滴加，以免激起柱顶层) 4) 装填完柱子后，用塑料滴管吸取PBS，沿管口四壁旋转着缓慢加入，使PBS过一遍Sephadex G-50柱子，而后将PBS注入Sephadex G-50顶部，以隔绝空气，以免在Sephadex G-50 柱内产生气泡，关闭末端活塞; 9. 过柱层析分离吸附柱(避光) 1) 打开柱子末端活塞，缓慢放出Sephadex G-50柱子顶层的PBS，待PBS液面刚与Sephadex G-50柱子端面相平时，立即关闭活塞; 注意不要使PBS液面低于Sephadex G-50柱子端面，以免混入空气而在柱子内产生气泡; 立即用塑料滴管吸取BSA-FITC荧光蛋白溶液，沿柱子四壁旋转，缓慢加入(全部加完)， 注:不要搅动Sephadex G-50，随时保持其端面水平; 2) 待BSA-FITC荧光蛋白溶液全部加完后，打开活塞，使 BSA-FITC荧光蛋白溶液流入Sephadex G-50柱子中; 当BSA-FITC荧光蛋白溶液的液面刚刚完全进入Sephadex G-50柱子时，立即用塑料滴管加入PBS(缓慢沿管子四壁旋转 加入)，在BSA-FITC蛋白溶液过Sephadex G-50柱子的过程中 注意随时添加PBS，确保Sephadex G-50端面不与空气接触; 3) 用烧杯承接最初的淋出液，3-5min后淡黄色的溶液便从柱 子顶部往下流，而后分作三段; 柱子下端(靠近活塞):BSA-FITC，淡黄色，流速较快; 柱子中间:空白无颜色部分，两者的过渡区域; 柱子上端:未与BSA反应的FITC(自由的FITC)，黄色，颜色较深，流速较慢; 4) 待下端淡黄色的BSA-FITC溶液的前沿进入活塞时，立即用45mL冻存管(锡箔纸避光)承接此 淋出液(此即BSA-FITC荧光蛋白溶液); 直至BSA-FITC溶液的尾部进入活塞口为止(也可再承接中间无颜色过渡区域部分的一半)。 Separate the unbound FITC from the conjugate by gel filtration using a fine-sized gel matrix with an exclusion limit of 20,000 to 50,000 (for globular proteins such as antibodies). With the column flow stopped, carefully layer the reaction mixture onto the top of the column. Then open the column, allowing the reaction mixture to flow into the column. Just as it all enters the column bed, carefully add PBS to the top of the column and connect to a buffer supply. Two bands will form on the column. The faster moving band, which is the conjugated protein, elutes first and can usually be seen under room light. The slower moving band is the unreacted (free) FITC and xylene cyanol and will elute only with subsequent PBS washes. 10. 计算Protein-FITC蛋白溶液中FITC与Protein的Mol比F/P 1) 打开“UV-7504紫外可见分光光度计”，设定两个波长，λ=280nm，λ=495nm;12 2) 取以准备好的两个石英比色皿C,D;C:3mL PBS;D:3mL BSA-FITC蛋白溶液; λ=280nm: 1 C:按“0ABS/100%T”清零(清零时用相应蛋白质溶液的溶剂); D:测BSA-FITC蛋白溶液在280nm处的吸光系数A(三次取平均值); 280 λ=495nm: 2 C:按“0ABS/100%T”调零; D:测BSA-FITC蛋白溶液在495nm处的吸光系数A(三次取平均值); 495 依相关公式计算FITC与BSA的Mol比。 A=0.694 (在[0.2,1.4]之间) A=0.829 280495%0.1C = (Mw* E)/(389*195); F/P = A*C / (A -0.35*A) 495280495%0.1C = (Mw* E)/(389*195) = (67000*0.619)/(389*195)=0.547; F/P = A*C / (A -0.35*A) = 0.547*0.829/ (0.694-0.35*0.829)=1.12;(不在[0.2,1.4]间， )495280495 3) 后处理: C:倒掉PBS，DIW冲洗，烘干; D:将BSA-FITC蛋白溶液取出，放回原来45mL冻溶管中，4?C暂存， 用SDS浸泡石英比色皿D，而后用DIW冲洗，烘干; The ratio of fluorescein to protein of the product can be estimated by measuring the absorbance at 495 nm and 280 nm. The F/P ratio should be between 0.3 and 1.0. Lower ratios will yield low signals; higher ratios will give high background. Determination of Fluorescein/Protein Molar Ration (F/P) The F/P molar ratio is defined as the ratio of moles of FITC to moles of protein in the conjugate. To determine this ratio, it is necessary to first determine the absorbance of the conjugate sample at 280 nm and then at 495 nm. Place the conjugate sample in a quartz cuvette. Read the absorbance of the conjugate sample at 280 nm and 495 nm. The absorbance reading of the conjugate sample should be between 0.2 and 1.4 at 280 nm. If the absorbance reading is outside this range, adjust the sample dilution accordingly. For FITC-IgG conjugates only: From the absorbance readings (A280 and A495) of the conjugate sample, calculate the F/P ratio of t he conjugate according to the equations: The protein concentration of the fluorescein-IgG conjugate is calculated from the following formula: Where 1.4 is the A280 of IgG from most species at a concentration of 1.0 mg/mL at pH 7.0. For other FITC-protein conjugates: When any protein other than IgG is conjugated to FITC, use the general formula below, substituting the appropriate values for the particular protein: C is a constant value given for a protein. MW is the molecular weight of the protein. 389 is the molecular weight of FITC. 0.1%195 is the absorption E of bound FITC at 490 nm at pH 13.0. (0.35 × A) is the correction factor due to the absorbance of FITC at 280 nm. 4950.1%E is the absorption at 280 nm of a protein at 1.0 mg/mL. Store the conjugate at 4 ?C in the column buffer in light-proof container. Sodium azide may be added as a preservative (final concentration 15 mM). If the protein concentration is low (< 1 mg/mL), bovine serum albumin (BSA) may be added to a final concentration of 1%. 11. Protein-FITC蛋白溶液进行超滤并测定其浓度: 1) 过完Sephadex G-50柱后，BSA-FITC蛋白溶液的浓度较低，不利于后续使用; 故应进行超滤以提高其浓度(不宜进行冷冻干燥)，并测定其具体浓度; 2) 测定浓度后分装于0.5mL离心管中，插于泡沫板上，置于纸盒内避光，-70?C保存。 0.1mL超滤后的BSA-FITC/PBS蛋白溶液(Cx) + 2.9mLPBS;(混合液浓度Cx/30) 测其吸光度:A=0.461， A=0.425; 280495 C =1.0mg/mL BSA/DIW， 0%0.1其吸光度:E =0.619; %0.1) = C / E , 故Cx=15.13mg/mL; 从而有:(Cx/30)/ (A -0.35*A4950280 12. 后处理 1) (1) 加入PBS，将Sephadex G-50中未与BSA反应的FITC冲洗下来，而后再用PBS过几次柱子，将淡黄色的FITC溶液彻底清洗干净; (2) 当柱子内还有PBS时，用洗耳球从柱子末端将Sephadex G-50吹落于烧杯中; (3) 再用0.2mol/L NaOH(2g，250mL DIW)浸泡过夜，使Sephadex G-50沉降，再换两次，每次10min; 而后用2-3倍柱体积的PBS泡洗3-4次，搅拌，使其充分沉降，每次10min; (4)加入100mL PBS，0.02%NaN(m/v;0.02g，100mL)，将其保存于4?冰箱中(重复使用);3