Lyophilization FAQs

1. Why is protein freeze-dried? What is the effect of freeze-drying on protein?

Proteins are sensitive to heat, and freeze-drying can preserve the activity of the majority of proteins. It improves protein stability, extends storage time, and reduces shipping costs. However, freeze-drying can also lead to the loss of the active portion of the protein and cause aggregation and denaturation issues. Nonetheless, these adverse effects can be minimized by incorporating protective agents such as stabilizers, additives, and excipients, and by carefully controlling various lyophilization conditions.

2. What is the general protectant? What kind of protectant do you usually add?

Commonly used protectant 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, the extension and aggregation of proteins during freeze-drying process; mannitol is also a universal applied protectant and fillers, which can reduce the aggregation of certain proteins after lyophilization. 

3. Why is the protein product barely visible in the tube?

Our protein products do not contain carrier protein or other additives (such as bovine serum albumin (BSA), human serum albumin (HSA) and sucrose, etc., and when lyophilized with the solution with the lowest salt content, they often cannot form A white grid structure, but a small amount of protein is deposited in the tube during the freeze-drying process, forming a thin or invisible transparent protein layer.

Reminder: Before opening the tube cap, we recommend that you quickly centrifuge for 20-30 seconds in a small centrifuge, so that the protein attached to the tube cap or the tube wall can be aggregated at the bottom of the tube. Our quality control procedures ensure that each tube contains the correct amount of protein, and although sometimes you can't see the protein powder, the amount of protein in the tube is still very precise.

4. How should I properly dissolve my recombinant protein product?

Step 1: Centrifuge the reagent tubes before opening the caps. The freeze-dried powder may drift and stick to the tube wall or cap during transportation, so before opening the plastic bottle cap, the freeze-dried powder should be collected to the bottom of the tube by centrifugation, so as to use a small volume The liquid can completely dissolve the lyophilized powder. Generally, it needs to be centrifuged at 3000-3500rpm for 5 minutes, and the effect is good.

Step 2: Resuspend to 0.1-1.0 mg/ml with sterile water, do not shake. This step is the dissolution step and is very important.

  1. Be sure to resuspend (or dissolve) the lyophilized powder in the recommended solution. The solubility of protein is related to many factors, the more important ones are pH value and ionic strength. The dissolving solution indicated on the product description is a liquid that can completely dissolve the cytokine or recombinant protein. If the pH value and ionic strength of the solution you use are inconsistent with those indicated in the instructions, many times the cytokine or recombinant protein cannot be completely dissolved or cannot be dissolved at all, so the prepared cytokine or recombinant protein must not be active enough or lost.
  2. Be sure to dissolve to the specified concentration. The protein can maintain good stability within a certain concentration range, which is the concentration range indicated on the instructions, generally 0.1-1.0 mg/ml. But this concentration range is different for different recombinant proteins. When the concentration is higher or lower than this range, the cytokine or protein will be unstable, that is, the phenomenon of decreased activity will easily occur. Secondly, above this concentration range, the maximum solubility concentration of the protein may be exceeded, that is, the protein cannot be completely dissolved. Furthermore, above or below this concentration range, the protein may aggregate, and the final result is that part of the protein is not dissolved, resulting in weakened protein activity.
  3. Must not oscillate (vortex). The oscillation mentioned here refers to the rapid oscillation with a vortex instrument. Use buffers that have been brought to room temperature as solvents. After adding buffer, cap the vial and gently invert the vial by hand or place the vial on a slow rocking shaker. Do this to ensure that the buffer reaches the entire inner wall of the bottle. Allow the bottle to sit at room temperature for at least 10 - 15 minutes before dispensing or using.

Step 3: The resuspension can be stored at 2-8°C for up to 1 week.

After the cytokine or recombinant protein is resuspended to the recommended concentration with the solution recommended in the instructions, the cytokine or recombinant protein can be placed at 2-8°C, that is, in the freezer of the refrigerator. Under these conditions, the activity of cytokines or recombinant proteins can be maintained for up to 1 week. This is sufficient for an experiment with a period of 5-7 days, such as induction of DC (dendritic cell) maturation. During this period, as long as a certain amount of cytokine or recombinant protein solution is drawn from the refrigerator each time and added to the culture system.

In fact, the concentration recommended in the instructions is relatively high for general experiments. Therefore, users usually further dilute the solution and store it at 4°C, and use it up within 1 week. If you want to dilute, you must follow the method of step 4 below, that is, you need to dilute with a solution containing carrier protein, otherwise the diluted cytokine or recombinant protein will easily adhere to the wall of the tube or bottle, making the solution in the solution The concentration of cytokines or recombinant proteins decreases, and the total activity of cytokines or recombinant proteins is greatly weakened.

Step 4: For long-term storage, it needs to be further diluted with a solution containing carrier protein (such as 0.1% BSA, or 10% FBS, or 5% HSA), and then aliquoted and frozen at -20°C to -80°C.

If an experiment cycle is longer than one week, or the prepared cytokine or recombinant protein cannot be used up at one time, we need to store the cytokine or recombinant protein for a long time. The method is: resuspend the resuspended cytokine or protein with a solution containing carrier protein, such as 0.1% BSA (bovine serum albumin), 10% FBS (fetal bovine serum), and 5% HSA (human serum albumin). The suspension was further diluted, and then subpackaged and frozen at -20°C to -80°C, that is, in the freezer of a conventional refrigerator or an ultra-low temperature freezer.

Be sure to further dilute the resuspension with a solution containing the carrier protein before aliquoting and freezing. The diluted cytokine or recombinant protein can be at any concentration, because a large amount of carrier protein can ensure that the low concentration cytokine or recombinant protein still maintains high stability.

That is, when doing serum-free culture or in vivo experiments on animals, the cytokines cannot contain animal or human proteins such as BSA, FBS or HSA. If you want to preserve cytokines or recombinant proteins for a long time, you can use trehalose (Trehalose) as a carrier. Dilute resuspended cytokines or recombinant proteins with a trehalose-containing solution, then aliquot and freeze.