Factor Xa Protease
目录号 : GC26287For Factor Xa, fusion protein cleavage is carried out at a w/w ratio of 1% the amount of fusion protein (e.g., 1 mg Factor Xa for a reaction containing 100 mg fusion protein).
Sample solution is provided at 25 µL, 10mM.
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Quality Control & SDS
- View current batch:
If necessary, concentrate the fusion protein to at least 1 mg/ml.
Do a pilot experiment with a small portion of your protein.
Example: Mix 20 µl fusion protein at 1 mg/ml, with 1 µl Factor Xa diluted to 200 µg/ml, or 0.2 ng Enterokinase
In a separate tube, place 5 µl fusion protein with no protease (mock digestion). Incubate the tubes at room temperature. At 2, 4, 8, and 24 hours, take 5 µl of the reaction, add 5 µl 2x SDS-PAGE Sample Buffer, and save at 4°C. Prepare a sample of 5 µl fusion protein + 5 µl 2X sample buffer (uncut fusion).
Boil the 6 samples for 5 minutes and run on an SDS-PAGE gel.
Scale the pilot experiment up for the portion of the fusion protein to be cleaved. Save at least a small sample of the uncut fusion as a reference.
Check for complete cleavage by SDS-PAGE.
Denaturing the Fusion Protein
1. Either dialyze the fusion against at least 10 volumes 20 mM Tris-HCl, 6 M guanidine hydrochloride, pH 7.4 for 4 hours, or add guanidine hydrochloride directly to the sample to give a final concentration of 6 M.
2. Dialyze against 100 volumes Column Buffer, 2 times at 4 hours each.
During refolding, one has to balance between two objectives. For the protease to cleave it must be present before the protein has completely refolded, so removing the denaturant quickly is desirable. However, when the denaturant is removed quickly some proteins will fail to refold properly and precipitate. Stepwise dialysis against buffer containing decreasing amounts of guanidine hydrochloride can prevent precipitation of the fusion protein; halving the guanidine concentration at each step is convenient, but cases where 0.1 M steps are necessary have been reported. However, if the fusion protein is able to refold into a protease-resistant conformation, it may be better to dialyze away the denaturant in one step and take the loss from precipitation in order to maximize the amount of cleavable fusion protein recovered.
Go to step 2 or 4 above, as appropriate.
For Factor Xa, fusion protein cleavage is carried out at a w/w ratio of 1% the amount of fusion protein (e.g., 1 mg Factor Xa for a reaction containing 100 mg fusion protein). The reaction mixture can be incubated for 3 hours to several days, at room temperature or 4°C. Depending on the particular fusion protein, the amount of protease can be adjusted within the range of 0.1–5.0%, to get an acceptable rate of cleavage. Factor Xa will cleave at non-canonical sites in some proteins; for some fusions, there is a correlation between instability of the protein of interest in and cleavage at additional sites (unpublished observations). Presumably this cleavage activity depends on the three dimensional conformation of the fusion protein. For fusions that are resistant to cleavage, two strategies can sometimes help. Inclusion of small amounts of SDS (0.005–0.05%) in the reaction appears to relax the fusion enough to allow for cleavage in some cases. The window of SDS concentrations that work can be small, so a pilot titration with different SDS concentrations is necessary. Another strategy that sometimes helps is to denature the fusion to render the protease site accessible to cleavage.
| Cas No. | SDF | ||
| 分子式 | 分子量 | 43 kDa | |
| 溶解度 | 储存条件 | Store at -20°C | |
| General tips | 请根据产品在不同溶剂中的溶解度选择合适的溶剂配制储备液；一旦配成溶液，请分装保存，避免反复冻融造成的产品失效。 储备液的保存方式和期限：-80°C 储存时，请在 6 个月内使用，-20°C 储存时，请在 1 个月内使用。 为了提高溶解度，请将管子加热至37℃，然后在超声波浴中震荡一段时间。 |
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| Shipping Condition | 评估样品解决方案：配备蓝冰进行发货。所有其他可用尺寸：配备RT，或根据请求配备蓝冰。 | ||
| 制备储备液 | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 23.2558 mL | 116.2791 mL | 232.5581 mL |
| 5 mM | 4.6512 mL | 23.2558 mL | 46.5116 mL |
| 10 mM | 2.3256 mL | 11.6279 mL | 23.2558 mL |
| 第一步：请输入基本实验信息（考虑到实验过程中的损耗，建议多配一只动物的药量） | ||||||||||
| 给药剂量 | mg/kg |  |
动物平均体重 | g | |
每只动物给药体积 | ul | |
动物数量 | 只 |
| 第二步：请输入动物体内配方组成（配方适用于不溶于水的药物；不同批次药物配方比例不同，请联系GLPBIO为您提供正确的澄清溶液配方） | ||||||||||
| % DMSO % % Tween 80 % saline | ||||||||||
| 计算重置 | ||||||||||
计算结果:
工作液浓度： mg/ml；
DMSO母液配制方法： mg 药物溶于 μL DMSO溶液（母液浓度 mg/mL，
体内配方配制方法：取 μL DMSO母液，加入 μL PEG300，混匀澄清后加入μL Tween 80，混匀澄清后加入 μL saline，混匀澄清。
1. 首先保证母液是澄清的；
2.
一定要按照顺序依次将溶剂加入，进行下一步操作之前必须保证上一步操作得到的是澄清的溶液，可采用涡旋、超声或水浴加热等物理方法助溶。
3. 以上所有助溶剂都可在 GlpBio 网站选购。