Q: 1. Why does protein need lyophilization? What influences do the lyophilization have on proteins?
A: The proteins are very sensitive to heat, protein lyophilization can make most of the activity retain, improve protein stability and extend the storage time, meanwhile reduce shipping costs.
• Lyophilization may cause partial loss of protein activity, aggregation and other degenerative problems. However, by adding protective agents (stabilizers, additives, excipients) and control ling various lyophilized conditions can reduce these negative effects as much as possible.
• Tips: Generally Cusabio provides lyophilized powder, they are very stable (at least one month) at room temperature. Nevertheless, we suggest to store at -20 ℃ upon receipt of our products, to ensure 100% activity of the protein.
Q: 2. Why should we add protective agent to the protein solution before lyophilization? What’s the general protective agent? Which kind of protective agent do you usually add?
A: The protective agent is used in the process of freeze-drying and storage to protect protein. Commonly used protective agents or stabilizers include saccharides, polyols, polymers, surfactants, some proteins and amino acids etc. We usually add 8% (mass ratio by volume) of trehalose and mannitol as lyoprotectant. Trehalose can significantly prevent the alter of the protein secondary structure and the extension and aggregation of proteins during freeze-drying process; mannitol is also a universal applied lyoprotectant and fillers, which can reduce the aggregation of certain proteins after lyophilization.
Tips: For the majority of proteins, they can be only stored at 4 ℃ for a short term (about one week) after resuspended. If you want a long-term preservation, first formulated as a diluent (which must contain a carrier protein, such as 0.1% BSA, 5% HSA, or 10% FBS), then repackage and freeze at -20℃ or -80℃. Be sure to avoid repeated freezing and thawing, because each freezing and thawing cycle will cause part of the protein inactivation.
Q: 3. Why our protein products are almost invisible in pipes?
A: Cusabio protein product does not contain carrier protein or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA) and sucrose, and when freeze-dring the lowest salt content solution often does not form a white grid structure, but a trace amount of protein deposit within the tube, forming a thin transparent or invisible protein layer.
Tips: Before opening the lid, we recommend to centrifuge in a small centrifuge for 20-30 seconds firstly, so aggregates the protein that attached to the wall or cap of the tube in the bottom of the tube. Our quality control steps ensures that the amount of protein contained in each tube is accurate, although sometimes you can’t see the protein powder, but the protein content in the tube is still very accurate.
Q: 4. How to determine species cross-reactivity of cytokines?
A: (1) Apart from a few exceptions, most human cytokines are active on mouse cells. (2) Many mouse cytokines may also have effect on human cells, however, the activity may be lower than the corresponding human cytokines. (3) One of the few human cytokines will be more active than corresponding mouse cytokines when acting on mouse cells, such as IL-7. (4) Interferon, GM-CSF, IL-3 and IL-4 and other cytokines are species-specific and almost have no activity on non-homologous cells. (5) In contrast, fibroblast growth factor (FGF) and neurotrophin are highly conserved and both have good activity on cells of different species.
Q: 5. How to properly dissolve recombinant protein product?
A: Step 1: Centrifugate the reagent tube before opening the cap. The lyophilized powder may drift and adsorb on the tube wall or the cap due to bump during the transportation. Before opening the plastic cap, collect the lyophilized powder to the tube bottom through centrifugation, in order to completely dissolve the lyophilized powder with a small volume of liquid. Generally, need to centrifugate for 5min at 3000-3500rpm to ensure a good effect.
Step 2: Resuspend in sterile water to 0.1-1.0 mg/ml with no oscillation. This step is the dissolving step and it’s very important.
1. Be sure to use the recommended solution to resuspend (or dissolve) the lyophilized powder. The solubility of protein is related to many factors, among them, pH and ionic strength are relatively important. The dissolving solution indicated in the product datasheet is the liquid which can completely dissolve the cytokine or recombinant protein. If the pH and ionic strength of the user’s solution are inconsistent with the product datasheet, in most case, this will cause that the cytokine or recombinant protein can’t be dissolved completely or even can’t be dissolved, then the prepared cytokine or recombinant protein will absolutely be lack or loss of activity.
2. Protein must be dissolved to the indicated concentration. Protein can maintain good stability within certain concentration range, which is indicated in the product datasheet, generally 0.1-1.0 mg / ml. However, the specific concentration range for different recombinant protein will be different. Above or below this concentration range, the cytokine or protein will be unstable, and it is very easy to appear the phenomenon of activity decline. Secondly, above this concentration range, it may exceed the maximum dissolved concentration of the protein, and the protein can’t be dissolved completely. Furthermore, above or below this concentration range, protein aggregation may occur, which will still result in part of protein undissolved and activity decline.
3. Must not oscillate (vortex). The oscillation mentioned here refers to the fast oscillation with vortex. Use the buffer stored at room temperature as the solvent. After adding the buffer, cover the bottle cap, gently flip the bottle by hand or place the bottle on a slowly rocking shaker, to guarantee that the buffer can touch the entire inner wall of the bottle. Place the bottle at room temperature for at least 10 – 15 minutes, then, make aliquots or use directly.
Step 3: This resuspension can be stored no more than one week at 2-8 ° C.
• Resuspend the cytokines or proteins with recommended buffer to the recommended concentration according to our manual, and then store at 2-8 ° C in the refrigerator, under this condition, the activity of the cytokines or proteins can maintain up to one week. So there is enough time to do a test during 5-7 days, such as the DC induced maturation test. During the experiment period, just remove a certain amount of cytokines or proteins from the refrigerator and add into the culture medium.
• In fact, the recommended concentration in our manual is a little high for usual tests. So the solution should be diluted and stored at 4 ° C, and then be used up within one week. For the dilution, please operate according to the step 4 below. Use the solution containing carrier protein to conduct dilution, otherwise, the diluted cytokines or proteins will adhere to the tube wall or bottle wall easily, which will cause the cytokines or proteins concentration decreasing, then weaken its activity sharply.
Step 4: As for long-term storage, the cytokines or proteins should be diluted further with the solution containing carrier protein (such as 0.1% BSA, 10% FBS, or 5% HAS), and then be stored at -20 ° C or -80 ° C.
• If an experiment is longer than one week, or the prepared cytokines or proteins can’t be used up for one time, and then long-term storage is needed. Method: Dilute the resuspended cytokines or proteins with solution containing carrier protein (such as 0.1% BSA, 10% FBS, or 5% HAS), then aliquot and store at -20°C or -80°C, conventional refrigerator freezer or ultra-low temperature refrigerator.
• Before aliquot and freeze, the resuspension should be diluted with the solution containing carrier protein. The cytokines or recombinant proteins can be diluted to any concentration, because large quantity of carrier proteins could keep the low-concentration cytokines or proteins still maintaining a high stability.
When performing the serum-free culture or animal experiment in vivo, the cytokines should contain no human or animal proteins such as BSA, FBS, HAS. As for the long-term storage, use Trehalose as the carrier, then dilute the resuspended cytokines or proteins with Trehalose solution, and then aliquot and store at -20°C or -80°C.