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      熒光FITC/CY3/5.5/7.5-NHS標記蛋白的實驗方法和步驟

      時間:2020-03-30 13:27:05       瀏覽:1226

      熒光FITC/CY3/5.5/7.5-NHS標記蛋白的實驗方法和步驟

      西安瑞禧生物科技有限公司是國內最大的活性基團熒光染料生產銷售商,我公司有提供像FITC-NHS、6-FAM NHS ester、5-TAMRA, SE、Cyanine3 NHS ester、Cyanine5 NHS ester、Cy7.5 NHS ester、ICG NHS ester、BDP 630/650 X NHS ester、Pyrene azide、BDP FL maleimide、Cyanine5-alkyne等不同產品,我們還可以提供像近紅外二區等特殊定制染料。

       

      西安瑞禧生物科技有限公司除了可以提供這些基礎染料還可以提供這些染料標記的蛋白產品包括有:FITC-BSA、BSA-TRITC、CY5.5-BSA、FITC-Streptavidin、CY5.5-Streptavidin、FITC-Concanavalin A、CY7.5-Con A、FITC-Dextran、FITC-Hyaluronate等不同產品。以及我們能提供各種熒光標記不同蛋白的定制服務。

       

      FITC/CY3/5.5/7.5-NHS/ICG-NHS/ATTO-NHS標記蛋白的實驗方法和步驟:

       

      (以下內容來自西安瑞禧生物科技有限公司,此文未經西安瑞禧生物允許不得私自轉發,違者必究)

       

      Fluorescein isothiocyanate (FITC,異硫氰酸熒光素) is widely used toattach a fluorescent label to proteins via the amine group. The isothiocyanategroup reacts with amino terminal and primary amines in proteins. It has been used forthe 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 forsolubility and solution appearance at 1 mg/mL in acetone to give a clear yellowsolution. It is soluble in anhydrous dimethyl sulfoxide (DMSO) at 5 mg/mL. Itis soluble in water at less than 0.1 mg/mL in water, at 20 mg/mL in ethanol andat 9 mg/mL in 2-methoxyethanol. Anorganic solvent for stock solution is advised, since FITC decomposes in water.FITC is diluted in basic buffer for coupling procedures immediately prior touse.

       

      Storage/Stability

      The products arelight-sensitive, and should be stored dry and in the dark at 2 °C to 8 °C.

       

      Procedure of LabelingProtein with FITC

      1.     Preparation before experiment:

      1)      100mL,0.1M PH=8.5SB緩沖液(0-5保存,且不超過一周,最好現配現用);

      2)      準備好A,B,C,D四個石英比色皿;

      3)      取一小容量瓶,事先用錫箔紙將其完全包住以避光;

      4)      用超純水注滿D=15mm,L=300mm柱層析分離吸附柱,過夜浸泡,同時檢測其是否漏液,第二天再用PBS浸泡7-8h;

      5)      DIW浸泡葡聚糖Sephadex G-50過夜(1gSephadex G-505mLDIW),使其溶脹,打開孔狀結構;

      而后將DIW倒掉,注入適量PBS(PH=7.4),用錫箔紙蓋好防灰,4保存;
      注:(1) Sephadex G-50量可以過多,過完柱層析分離吸附柱后剩余部分可加入  0.02%(m/v)NaN3,防止細菌,保存于4冰箱中;

      (2) Sephadex G-50第一次配好后可以反復使用多次,但應做相應后處理(見后);

      (3) Sephadex G-50分離范圍:1000-30,000(分子量),

       

      2.      計算Protein中游離-NH2,配制1.0mg/mL Protein/SB蛋白溶液:
      BSA, Fraktion V       Mw=67000Da,           Arg 26;  lys 60;

      20mg BSA;            2mLSB(0.1M PH=8.5);
      游離-NH2n1=20×0.001g×(26+60)/(67000g.mol-1) =2.57×10-5mol;
      溶于小容量瓶中(外包錫箔紙避光處理),加攪拌子攪拌,使其完全溶解;

      注:組成蛋白質的20中氨基酸中,僅Arg,lys在側鏈上有游離的-NH2。

       

      3. 求算FITC的量:

      5.8mg FITC,5.8mLDMSO(干燥),溶于5mL凍溶管中;

      注:(1) 配好后分裝于0.5mL離心管中,-69°C保存,之后可以直接使用。

      (2) FITC的量應適中,以使蛋白質中所有游離的-NH2均與FITC反應,

      FITC量較少 未反應-NH2較多 → F/P低;

      FITC量過多 →  F/P高;

      (2) BSA,取已配好的150uL 1.0mg/mLFITC/DMSO,

      FITCn2=0.15×1.0×0.001/389.4 mol =0.385×10-6mol

       

      Dissolve the FITC in anhydrousDMSO at 1.0mg/mL.

      Note: This should be prepared fresh for each labeling reaction.

       

      4. FITC標記Protein反應:

      用移液器每次取10ul,間隔、緩慢加入BSA/SB蛋白溶液中;
       
      全部加完后,攪拌、混合一會,轉入4,黑暗中反應8h(冰箱內);

      注:加入FITC的過程中應注意避光。

       

      For each 1 mL ofprotein solution, add 50 mL of FITC solution, very slowly in 5 mL aliquotswhile gently and continuously stirring the protein solution.

      After all the requiredamount of FITC solution has been added, incubate the reaction in the dark for 8hours at 4 °C.

       

      5. 1.0mg/mL Protein/SB蛋白溶液在280nm處的吸光系數E%0.1。

      配制3mL 1.0mg/mL BSA/SB蛋白溶液(5mL凍溶管中),用“UV-7504紫外可見分光光度計測其在280nm處的吸光系數E%0.1。
      A
      3mLSB,按“0ABS/100%T”清零(清零時用相應蛋白質溶液的溶劑);
      B
      3mL1.0mg/mL BSA/SB蛋白溶液,測其在280nm處的吸光系數E%0.1;

      后處理:A:倒掉3mL SB,DIW沖洗,烘干;
      B
      :將BSA/SB蛋白溶液取出,放回原來5mL凍溶管中,4°C保存,
        
      SDS浸泡石英比色皿B,而后用DIW沖洗,烘干;

       

      6. NH4Cl終止反應

      Add NH4Cl to a final concentration of 50 mM, mix for a whilethen incubate for 2 hours at 4 °C.

      NH4Clm1=50×0.001mol/L×(2.0+0.15)×0.001L×53.49g/mol=5.75×10-3g=0.00575g;

       

      7. Add xylene cyanolto a final concentration of 0.1%(m/v) and glycerol to 5%(m/v).

      xylene cyanol 0.0042g,顯色作用,使過柱時蛋白質與FITC易于分辨;
      (
      本實驗未加,最下層為蛋白質)

      glycerol 甘油 m2= (2.0+0.15) mL×5%=0.1075g (2mL離心管稱取)

      攪拌,混合5min左右;

       

      8. 裝填柱層析分離吸附柱

      1) 45mL凍存管,用錫箔紙包好以避光;

      2) 濕法裝柱:(裝填過程中確保PBS液面比Sephadex G-50高,以防混入空氣而產生氣泡)

      先在柱子內注入3/4左右的PBS,而后加入Sephadex G-50/PBS溶液;分批次、緩慢加入柱子內,最后用塑料滴管吸取,沿著柱子四壁旋轉,逐步、緩慢地加入,裝填至近滿,留下4-5cm的空間以待注入BSA-FITC熒光蛋白溶液;

      3) 在裝填的整個過程中打開活塞(淋出液用大燒杯承接),同時用玻璃棒+橡膠塞從下到上輕輕敲打柱子,使Sephadex G-50裝填緊密,注意使Sephadex G-50柱子內不得有氣泡,不得有斷層,使柱子豎直,確保頂部Sephadex G-50端面水平;

      (故滴加SephadexG-50時應旋轉加入,不得直接滴加,以免激起柱頂層)

      4) 裝填完柱子后,用塑料滴管吸取PBS,沿管口四壁旋轉著緩慢加入,使PBS過一遍SephadexG-50柱子,而后將PBS注入SephadexG-50頂部,以隔絕空氣,以免在Sephadex G-50 柱內產生氣泡,關閉末端活塞;

       

      9. 柱層析分離吸附柱(避光)

      1) 打開柱子末端活塞,緩慢放出Sephadex G-50柱子頂層的PBS,待PBS液面剛與Sephadex G-50柱子端面相平時,立即關閉活塞;

      注意不要使PBS液面低于Sephadex G-50柱子端面,以免混入空氣而在柱子內產生氣泡;

      立即用塑料滴管吸取BSA-FITC熒光蛋白溶液,沿柱子四壁旋轉,緩慢加入(全部加完),

      注:不要攪動Sephadex G-50,隨時保持其端面水平;

      2) BSA-FITC熒光蛋白溶液全部加完后,打開活塞,使BSA-FITC熒光蛋白溶液流入Sephadex G-50柱子中;

      BSA-FITC熒光蛋白溶液的液面剛剛完全進入Sephadex G-50柱子時,立即用塑料滴管加入PBS(緩慢沿管子四壁旋轉加入),在BSA-FITC蛋白溶液過Sephadex G-50柱子的過程中注意隨時添加PBS,確保Sephadex G-50端面不與空氣接觸;

      3) 用燒杯承接最初的淋出液,3-5min后淡黃色的溶液便從柱子頂部往下流,而后分作三段;

      柱子下端(靠近活塞)BSA-FITC,淡黃色,流速較快;

      柱子中間:空白無顏色部分,兩者的過渡區域;

      柱子上端:未與BSA反應的FITC(自由的FITC),黃色,顏色較深,流速較慢;

      4) 待下端淡黃色的BSA-FITC溶液的前沿進入活塞時,立即用45mL凍存管(錫箔紙避光)承接此淋出液(此即BSA-FITC熒光蛋白溶液);

      直至BSA-FITC溶液的尾部進入活塞口為止(也可再承接中間無顏色過渡區域部分的一半)。

       

      Separate the unboundFITC from the conjugate by gel filtration using a fine-sized gel matrix with anexclusion 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 topof the column. Then open the column, allowing the reaction mixture to flow intothe column. Just as it all enters the column bed, carefully add PBS to the topof the column and connect to a buffer supply. Two bands will form on thecolumn. The faster moving band, which is the conjugated protein, elutes firstand can usually be seen under room light. The slower moving band is theunreacted (free) FITC and xylene cyanol and will elute only with subsequent PBSwashes.

       

      10. 計算Protein-FITC蛋白溶液中FITCProteinMolF/P

      1) 打開“UV-7504紫外可見分光光度計,設定兩個波長,λ1=280nm,λ2=495nm;

      2) 取以準備好的兩個石英比色皿C,D;C3mL PBS;D3mL BSA-FITC蛋白溶液;

      λ1=280nm

      C:按“0ABS/100%T”清零(清零時用相應蛋白質溶液的溶劑);
      D
      :測BSA-FITC蛋白溶液在280nm處的吸光系數A280(三次取平均值);

      λ2=495nm

      C:按“0ABS/100%T”調零;
      D
      :測BSA-FITC蛋白溶液在495nm處的吸光系數A495(三次取平均值);

      依相關公式計算FITCBSAMol比。

      A280=0.694 ([0.2,1.4]之間)         A495=0.829

      C = (Mw*E%0.1)/(389*195);          F/P = A495*C/ (A280 -0.35*A495)

      C = (Mw* E%0.1)/(389*195) = (67000*0.619)/(389*195)=0.547;

      F/P = A495*C / (A280 -0.35*A495) =0.547*0.829/ (0.694-0.35*0.829)=1.12;(不在[0.2,1.4]間, )

      3) 后處理:

      C:倒掉PBS,DIW沖洗,烘干;
      D
      :將BSA-FITC蛋白溶液取出,放回原來45mL凍溶管中,4°C暫存,
        
      SDS浸泡石英比色皿D,而后用DIW沖洗,烘干;

       

      The ratio offluorescein to protein of the product can be estimated by measuring the absorbanceat 495 nm and 280 nm. The F/P ratio should be between 0.3 and 1.0. Lower ratioswill yield low signals; higher ratios will give high background.

      Determination ofFluorescein/Protein Molar Ration (F/P)

      The F/P molar ratio isdefined as the ratio of moles of FITC to moles of protein in the conjugate. Todetermine this ratio, it is necessary to first determine the absorbance of theconjugate sample at 280 nm and then at 495 nm.

      Place the conjugatesample in a quartz cuvette. Read the absorbance of the conjugate sample at 280nm and 495 nm. The absorbance reading of the conjugate sample should be between0.2 and 1.4 at 280 nm. If the absorbance reading is outside this range, adjustthe sample dilution accordingly.

       

      For FITC-IgG conjugates only:

      From the absorbancereadings (A280 and A495) of the conjugate sample, calculate the F/P ratio ofthe conjugate according to the equations:

      The proteinconcentration of the fluorescein-IgG conjugate is calculated from the followingformula:

      Where 1.4 is the A280of IgG from most species at a concentration of 1.0 mg/mL at pH 7.0.

       

      For other FITC-protein conjugates:

      When any protein otherthan IgG is conjugated to FITC, use the general formula below, substituting theappropriate 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.

      195 is the absorption E0.1% of bound FITC at 490 nm at pH 13.0.

      (0.35 × A495) is the correction factor due to the absorbance ofFITC at 280 nm.

      E0.1% 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 theprotein concentration is low (< 1 mg/mL), bovine serum albumin (BSA) may beadded to a final concentration of 1%.

       

      11. Protein-FITC蛋白溶液進行超濾并測定其濃度:

      1) 過完Sephadex G-50柱后,BSA-FITC蛋白溶液的濃度較低,不利于后續使用;

         故應進行超濾以提高其濃度(不宜進行冷凍干燥),并測定其具體濃度;

      2) 測定濃度后分裝于0.5mL離心管中,插于泡沫板上,置于紙盒內避光,-70°C保存。

         0.1mL超濾后的BSA-FITC/PBS蛋白溶液(Cx)  + 2.9mLPBS;(混合液濃度Cx/30)

           測其吸光度:A280=0.461,         A495=0.425;

         C0 =1.0mg/mL BSA/DIW,

      其吸光度:E%0.1=0.619;

         從而有:(Cx/30)/ (A280 -0.35*A495)= C0 / E%0.1 ,    Cx=15.13mg/mL;

       

      12. 后處理

      1) (1) 加入PBS,將Sephadex G-50中未與BSA反應的FITC沖洗下來,而后再用PBS過幾次柱子,將淡黃色的FITC溶液徹底清洗干凈;

      (2) 當柱子內還有PBS時,用洗耳球從柱子末端將Sephadex G-50吹落于燒杯中;

      (3) 再用0.2mol/L NaOH(2g,250mL DIW)浸泡過夜,使Sephadex G-50沉降,再換兩次,每次10min;

         而后用2-3倍柱體積的PBS泡洗3-4次,攪拌,使其充分沉降,每次10min;

      (4)加入100mL PBS,0.02%NaN3(m/v;0.02g,100mL),將其保存于4冰箱中(重復使用);

      2) 若后續需標記另一種蛋白,則柱子應用2%SDS浸泡過夜,而后用PBS,DIW清洗、烘干。

       



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