Q: How to reconstitute and store recommbinant proteins?
A: CHAMOT's recommbinant proteins are lyophilized powder, these products can be stored at -20℃ or -80℃ for 12 months in lyophilized state from date of receipt.
Upon receipt of the product, it is strongly recommended to centrifuge the product before opening, which can allow proteins that may attached to the cap or wall due to transport to accumulate at the bottom of the tube.
1. Centrifugation: Low speed centrifugation (3000-3500 rpm) for 5 minutes.
2. Dissolve:
Step 1: Dissolve with the recommended buffer, and the initial concentration is in the range of 0.1-1.0 mg/ml. No vortex! Since violent vortex will break the chemical bond of protein, resulting in protein inactivation. In order to ensure the dissolution complete ly, shake gently after adding appropriate solution or blow gently with a pipette for several times, and keep it at room temperature for about 20 min until the proteins are completely dissolved.
Step 2: Dilution: After initial reconstitution, it can be further diluted according to the needs of experiment. The dilution buffer such as medium or PBS can be used, which must contain carrier proteins or stabilizer such as BSA, FBS, HSA or trehalose.
3. Extended storage: the volume of the working aliquots is preferably for one experiment, and is at least 20ul,which can avoid protein loss caused by adsorption. Avoid repeated freeze/thaw cycles. Any freeze-thaw cycle will decrease the protein's activity.
* The protein can be stored at 4℃ for 1 week after initial reconstitution.For long-term storage, it is necessary to add a solution containing carrier protein or stabilizer for further dilution and then divide protein into several vials at -20℃ or -80℃, which can be stored for 3-6 months.
Q: Is it OK if the initial concentrition is out of the recommended range of the concentration?
A: Not recommended. Too high or too low concentration can lead to protein instability, and decrease the activity. Above this concentration range, the maximum dissolved concentration of the protein may be exceeded, and the protein can not be dissolved completely, which will affect protein's activity. In addition, above or below this concentration range, protein aggregation may occur, and will cause partially undissolved protein, resulting in decrase of protein's activity.
Q: What are the carrier proteins?
A: 0.1%BSA, 10%FBS(fetal bovine serum), 5%HSA(human serum albumin), 5% trehalose, choose one of four. 5% trehalose is recommended for serum-free experimental culture.
Q: What does a solution containing carrier protein mean? Is water OK?
A: In order to maintain a certain pH and salt concentration to avoid protein instability, water is not recommended. The solution is usually recommended with a neutral pH and a certain buffer capacity, such as PBS, medium (RPMI1640, DMEM, etc.).
Q: Some people will freeze the initial reconstitution directly at -20℃ to -80℃ without any carrier protein or stabilizer. Is this OK?
A: No. The plastic tube wall has a good adsorption effect on most proteins, which means that the proteins in the solution are easy to adhere to the tube wall. After adhesion, the protein is difficult to separate from the tube wall.
The main function of carrier proteins, such as 0.1% BSA, 10% FBS, 5% HSA and 5% trehalose, is to block the protein-binding sites on the plastic tube wall so that recombinant proteins do not adhere to the tube wall. If the lyophilized recombinant protein powder is reconstituted with the recommended solution, and does not further dilute in a buffer containing carrier protein, while it is directly frozen, most of the proteins in the solution will adhere to the tube wall, resulting in a significant reduction in the concentration of actually dissolved recombinant proteins. The final manifestation is a decrease of protein's activity. In addition, the carrier protein can also maintain the stability of the protein at low temperature, so that it can maintain activity.
Therefore, it is necessary to further dilute the reconstitution with a buffer containing carrier protein before extended long-term storage.
