Factor Xa Protease |
Catalog No.GC26287
One-Click Copy Product Info
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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).
Products are for research use only. Not for human use. We do not sell to patients.
Sample solution is provided at 25 µL, 10mM.
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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fusions, there is a correlation between instability of the protein of interest in and cleavage at additional sites (unpublished observations).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.
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.
| Cas No. | SDF | ||
| Formula | M.Wt | 43 kDa | |
| Solubility | Storage | Store at -20°C | |
| General tips | Please select the appropriate solvent to prepare the stock solution according to the
solubility of the product in different solvents; once the solution is prepared, please store it in
separate packages to avoid product failure caused by repeated freezing and thawing.Storage method
and period of the stock solution: When stored at -80°C, please use it within 6 months; when stored
at -20°C, please use it within 1 month. To increase solubility, heat the tube to 37°C and then oscillate in an ultrasonic bath for some time. |
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| Shipping Condition | Evaluation sample solution: shipped with blue ice. All other sizes available: with RT, or with Blue Ice upon request. | ||
| Prepare stock solution | |||
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1 mg | 5 mg | 10 mg |
| 1 mM | 23.3 μL | 116.3 μL | 232.6 μL |
| 5 mM | 4.7 μL | 23.3 μL | 46.5 μL |
| 10 mM | 2.3 μL | 11.6 μL | 23.3 μL |
Step 1: Enter information below (Recommended: An additional animal making an allowance for loss during the experiment)
g
μL
Step 2: Enter the in vivo formulation (This is only the calculator, not formulation. Please contact us first if there is no in vivo formulation at the solubility Section.)
Calculation results:
Working concentration: mg/ml;
Method for preparing DMSO master liquid: mg drug pre-dissolved in μL DMSO ( Master liquid concentration mg/mL, Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug. )
Method for preparing in vivo formulation: Take μL DMSO master liquid, next addμL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL saline, mix and clarify.
Method for preparing in vivo formulation: Take μL DMSO master liquid, next add μL Corn oil, mix and clarify.
Note: 1. Please make sure the liquid is clear before adding the next solvent.
2. Be sure to add the solvent(s) in order. You must ensure that the solution obtained, in the previous addition, is a clear solution before proceeding to add the next solvent. Physical methods such as vortex, ultrasound or hot water bath can be used to aid dissolving.
3. All of the above co-solvents are available for purchase on the GlpBio website.
Quality Control & SDS
- View current batch:
- Appearance: A liquid
- COA (Certificate of Analysis)
- SDS (Safety Data Sheet)
- Datasheet
Average Rating: 5 (Based on Reviews and 30 reference(s) in Google Scholar.)