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荧光FITC/CY3/5.5/7.5-NHS标记蛋白的实验方法和步骤

时间:2020-03-30 13:27:05       浏览:145

荧光FITC/CY3/5.5/7.5-NHS标记蛋白的实验方法和步骤

西安瑞禧生物科技有限公司是国内最大的活性基团荧光染料生产销售商,我公司有提供像FITC-NHS6-FAM NHS ester5-TAMRA, SECyanine3 NHS esterCyanine5 NHS esterCy7.5 NHS esterICG NHS esterBDP 630/650 X NHS esterPyrene azideBDP FL maleimideCyanine5-alkyne等不同产品,我们还可以提供像近红外二区等特殊定制染料。

 

西安瑞禧生物科技有限公司除了可以提供这些基础染料还可以提供这些染料标记的蛋白产品包括有:FITC-BSABSA-TRITCCY5.5-BSAFITC-StreptavidinCY5.5-StreptavidinFITC-Concanavalin ACY7.5-Con AFITC-DextranFITC-Hyaluronate等不同产品。以及我们能提供各种荧光标记不同蛋白的定制服务。

 

FITC/CY3/5.5/7.5-NHS/ICG-NHS/ATTO-NHS标记蛋白的实验方法和步骤:

 

(以下内容来自西安瑞禧生物科技有限公司,此文未经西安瑞禧生物允许不得私自转发,违者必究)

 

Fluorescein isothiocyanate (FITC,异硫氰酸荧光素) 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)      100mL0.1M PH=8.5SB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=15mmL=300mm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)      DIW浸泡葡聚糖Sephadex G-50过夜(1gSephadex G-505mLDIW),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-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)
游离-NH2n1=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Arglys在侧链上有游离的-NH2

 

3. 求算FITC的量:

5.8mg FITC5.8mLDMSO(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-NH2均与FITC反应,

FITC量较少 未反应-NH2较多 → F/P低;

FITC量过多 →  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 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处的吸光系数E%0.1

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测其在280nm处的吸光系数E%0.1
A
3mLSB,按“0ABS/100%T”清零(清零时用相应蛋白质溶液的溶剂)
B
3mL1.0mg/mL BSA/SB蛋白溶液,测其在280nm处的吸光系数E%0.1

后处理:A:倒掉3mL SBDIW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,
  
SDS浸泡石英比色皿B,而后用DIW冲洗,烘干;

 

6. NH4Cl终止反应

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,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 m2= (2.0+0.15) mL×5%=0.1075g (2mL离心管称取)

搅拌,混合5min左右;

 

8. 装填柱层析分离吸附柱

1) 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SephadexG-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SephadexG-50柱子,而后将PBS注入SephadexG-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. 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) 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;

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 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. 计算Protein-FITC蛋白溶液中FITCProteinMolF/P

1) 打开“UV-7504紫外可见分光光度计,设定两个波长,λ1=280nmλ2=495nm

2) 取以准备好的两个石英比色皿C,DC3mL PBSD3mL BSA-FITC蛋白溶液;

λ1=280nm

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A280(三次取平均值)

λ2=495nm

C:按“0ABS/100%T”调零;
D
:测BSA-FITC蛋白溶液在495nm处的吸光系数A495(三次取平均值)

依相关公式计算FITCBSAMol比。

A280=0.694 ([0.2,1.4]之间)         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]间, )

3) 后处理:

C:倒掉PBSDIW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,
  
SDS浸泡石英比色皿D,而后用DIW冲洗,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:A280=0.461         A495=0.425

   C0 =1.0mg/mL BSA/DIW

其吸光度:E%0.1=0.619

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

 

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(2g250mL 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 PBS0.02%NaN3(m/v0.02g100mL),将其保存于4冰箱中(重复使用)

2) 若后续需标记另一种蛋白,则柱子应用2%SDS浸泡过夜,而后用PBSDIW清洗、烘干。

 



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