PeptiTagTM Biotin - PEPTIDE LABELING KIT 生物素标记试剂盒
Introduction说明书
PeptiTag™生物素标记-是一种简单有效的利用生物素对单肽进行标记的试剂盒。试剂盒中的生物素-XP*会与N-末端上重要的胺基或赖氨酸侧链反应，在生物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，由于生物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，合成的肽链一般与生物素化试剂结合性较高，生物素-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
• 生物素-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
* Biotin-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. Biotinylation生物素化
1. Weight 0.5-1 mg of the Biotin-XP reagent in a 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 of (5-10µl) DMSO
(Dimethylsulfoxide) or reaction solution. Add 50µl reaction solution.
3. Calculate the amount of Biotin 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 400 x Biotin solution volume
Biotin mg x Peptide MW
4. Add X µl of Biotin 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 biotinylated 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 Biotin, and significantly reduces nonspecific interactions caused by it.
* Hydrophobic peptides that do not dissolve in water solutions are ready to use at the end of this step.
* The pellet should be dried completely – leftovers of precipitation solution interfire with peptide dissolving in
separation solution.
Part 2: Column Separation*层析
1. Dissolve the peptide in 0.1-0.2ml of Separation Solution.
2. Resuspend the Separation Gel before use.
3. Transfer 0.2-0.4ml of suspended Gel into the column. Place the column into a collection tube and centrifuge for
1 minute at 500 G to remove the solution.
4. Remove the spin column from the collection tube and discard the flow-through solution.
5. Wash the column once with 0.2ml Separation Solution (add solution to the column, centrifuge, and discard the
flow-through solution.
6. Add the dissolved peptide to the top of the gel in the spin column. Centrifuge the tube for 1 minute at 500 x g.
The flow-through contains your labeled peptide.
7. Store aliquots of your peptide at –20OC to –70OC.
* Column separation should be used only for water-soluble peptides that are readily soluble in Separation Solution.
* Some peptides change their properties upon labeling and become non soluble or slightly soluble in water – and can not
be separated with the use of the spin column.
MODIFIED PROTOCOL FOR LABELING PEPTIDES ALREADY DISSOLVED IN WATER
A. Biotinylation生物素化
1. Weight 0.2-0.5mg of the Biotin-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 Biotin 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.
* The pellet should be dried completely – leftovers of precipitation solution interfire with peptide dissolving in
separation solution.
Part 2: Column Separation*层析
1. Dissolve the peptide in 0.1-0.2ml of Separation Solution.
2. Resuspend the Separation Gel before use.
3. Transfer 0.2-0.4ml of suspended Gel into the column. Place the column into a collection tube and centrifuge for
1 minute at 500 G to remove the solution.
4. Remove the spin column from the collection tube and discard the flow-through solution.
5. Wash the column once with 0.2ml Separation Solution (add solution to the column, centrifuge, and discard the
flow-through solution.
6. Add the dissolved peptide to the top of the gel in the spin column. Centrifuge the tube for 1 minute at 500 x g.
The flow-through contains your labeled peptide.
7. Store aliquots of your peptide at –20OC to –70OC.
* Column separation should be used only for water-soluble peptides that are readily soluble in Separation Solution.
* Some peptides change their properties upon labeling and become non soluble or slightly soluble in water – and can not
be separated with the use of the spin column.
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. Biotinylation生物素化
1. Weight 0.2-0.5mg of the Biotin-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 Biotin 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.
