PeptiTagTM Fluorescein - PEPTIDE LABELING KIT荧光素标记试剂盒
Introduction说明
PeptiTag™荧光素标记-是一种简单有效的利用荧光素对单肽进行标记的试剂盒。试剂盒中的荧光素-XP*会与N-末端上重要的胺基或赖氨酸侧链反应，在荧光素与氨基酸之前形成共价氨基化合物。这种链接稳定牢固，不会在贮存过程中丢失标记物
再则，由于荧光素自由基是从生物素化单肽中分离出来的，可以在标记过程中从很大程度上降低非特异性反应；
这种试剂盒用于标记合成的肽链效果特别好，合成的肽链一般与标记性试剂结合性较高，荧光素-XP*能与主链上
的胺基反应，形成稳定的氨基化合物。
Kit Components试剂盒成份
•荧光素-XP*试剂
• 反应溶液
• 终止溶液
• 催化剂
• 分离胶
• 分离溶液
• 旋转柱
• 聚丙稀微型离心管
* 荧光素-XP是一种新型专利生物化试剂
IMPORTANT NOTES:注意事项：
•所有的试剂盒在使用前必需在4度条件中保存“不要冻存”
• 要想得到最佳效果就要使用干粉状的/无水蛋白肽链
• 若要延长生物素化单肽的贮存时间，则可以冻干它。
• 在使用已经溶解的肽链时，请确保溶液中不含其它胺类类杂质，如：铵，tris,谷胱甘肽，咪唑，糖胶。
如果溶液中含有这类物质，那么必须把它们层析或分离掉。
STANDARD PROTOCOL标准操作
A. Peptide Labeling单肽标记
1. Weight 0.5-1 mg of Fluorescein-XP reagent in 1.5ml polypropylene tube, and dissolve in 50µl-100µl reaction
solution (final concentration of 10mg/ml).
2. Weight 1-2 mg of your peptide in 2ml polypropylene tube, and dissolve in minimal amount (5-10µl) DMSO
(Dimethylsulfoxide) or reaction solution. Add 50µl reaction solution.
3. Calculate the amount of Fluorescein solution that should be added to the peptide according to the formula (molar
ratio from 2 to 6 can be applied)
X = molar ratio x Peptide mg x 500 x total solution volume (µl)
Fluorescein mg x Peptide MW
4. Add X µl of Fluorescein solution to the peptide solution. Mix by vortex.
5. Incubate for 2-3* hours at room temperature.
6. Add 5 µl stop solution. Mix by vortex.
* High molar ratio (6) is more time saving and efficient then low molar ratio (2).
* For low molar ratio and difficult peptides, incubation of 16-24 hours is recommended to achive complete labeling of the
peptide.
B. Purification纯化
Part 1: Precipitation*催化
1. Add 1ml of ice cold Precipitation Solution to the reaction mix, vortex. The precipitate that appears, contain the
labeled peptide.
2. Cool the tube on ice for 15-20 minutes, for improved precipitation of the product.
3. Centrifuge the tube for 1 minute at 3000 G, pellet appears in the bottom of the tube. Carefully remove the
solution, the pellet contains the labeled peptide.
4. Add 0.2ml ice cold Precipitation Solution to the pellet, and suspend by vortex.
5. Centrifuge the tube for additional 1 minute at 3000 g, and carefully remove the solution, try to remove all solution
residuals, dry* the pellet on air.
* This step removes most of the free Fluorescein, and significantly reduces nonspecific interactions caused by it.
l Hydrophobic peptides that do not dissolve in water solutions are ready to use at the end of this step
MODIFIED PROTOCOL FOR LABELING PEPTIDES ALREADY DISSOLVED IN WATER
.对已溶解在水中的肽链进行标记的改进手册
A. Peptide Labeling单肽标记
1. Weight 0.2-0.5mg of Fluorescein-XP reagent in 2ml polypropylene tube, and dissolve in 50µl -100µl reaction
solution (final concentration of 5mg/ml).
2. Add 5-10µl of the water-dissolved peptide to the Fluorescein reaction solution (dissolved peptide solution should
be less than 10% of the reaction mixture). Mix by vortex.
3. Incubate for 2-3 hours at room temperature.
4. Add 5 µl stop solution. Mix by vortex.
B. Purification纯化
Should be carried out as described before Part I and Part II.
