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Common problems of Abbkine recombinant protein dissolution

Date:2021-01-19 Views:109

The correct way to dissolve freeze-dried powder:

Step 1: Centrifuge before opening the cover.

During transportation, the freeze-dried powder will adhere to the tube wall or the tube cover  due to external forces. Before opening the cover, the freeze-dried powder will be collected at the bottom of the tube by centrifugal operation to avoid loss and waste. Centrifuge at 10000-12000rpm, 30s or 3000-3500rpm, 5min.

Step 2: After centrifugation, add the provided buffer to the freeze-dried powder, mix gently with a pipette tip, and resuspend to a concentration of not less than 100 ug/ml. (For example, the recombinant protein with the specification of 100 ug should be dissolved by adding 1 ml of buffer).

A: Use the provided buffer to resuspend the freeze-dried powder.

The pH and ionic strength of the resuspension solution have a very important impact on the solubility of the recombinant protein; If the buffer used is not suitable, it is likely that the freeze-dried powder cannot be completely dissolved or  insolubilized, which will eventually lead to insufficient or loss of activity of cytokines or recombinant proteins after dissolution.

B: Dissolve to the specified concentration.

According to the instructions in the manual, diluting the  freeze-dried powder to the specified concentration range is conducive to maintaining the good stability of cytokines or recombinant proteins. If it is higher or lower than the specified concentration range, on the one hand, the activity of cytokines or recombinant protein may decrease; On the other hand, proteins may aggregate, resulting in partial protein insolubilized and weakened activity.

Step 3: Cytokines or recombinant protein solution directly dissolved in the buffer can be stored for up to 1 week at 2-8℃.For the experiment with short period (no more than 7 days), the cytokine or recombinant protein solution stored under this condition can be directly added to the culture system . If the used concentration of the experimental conditions is lower than the concentration of the solution after reconstitution, please dilute with a solution containing carrier protein. If the diluent does not contain carrier protein,  it is easy to cause cytokine or recombinant protein to adhere to the tube wall or bottle wall , which will reduce the concentration of cytokines or recombinant proteins in the solution and further weaken the activity of cytokines or recombinant proteins. For long term storage it is recommended to add a carrier protein (0.1% HSA or BSA). Please prevent freeze-thaw cycles.

Step 4: For long-term storage, it needs to be further diluted with a solution containing carrier protein (0.1% BSA or HSA),  then stored separately and frozen at -20℃ to -80℃.

Note: The freeze-dried powder dissolved in the provided reconstituted buffer cannot be directly frozen at -20 to -80 ℃. Because the plastic tube wall has a good adsorption effect on the protein, it will cause the cytokine or recombinant protein to adhere to the tube wall and it is not easy to separate, which will cause the actual concentration of the cytokine or recombinant protein in the solution to be low, and ultimately lead to a decrease in its activity. And that carry protein can seal the protein binding site on the plastic pipe wall in advance, so that cytokine or recombinant protein can not adhere to the tube wall. Therefore, for long-term storage, before aliquoting and freezing, be sure to further dilute it with a solution containing carrier protein.

 

Frequently asked questions:

  1. Can Vortexingmachine be used to help freeze-dried powder dissolve fully?

In the process of dissolution, do not use Vortexingmachine to shake quickly, otherwise it will affect the activity of cytokines. It is recommended to gently blow and mix with a gun head or to mix up and down gently.

 

  1. According to the normal steps, what should we do if there is no complete dissolution in the dissolution process?

In general, the freeze-dried powder is very easy to dissolve, and it can be completely dissolved by gently blowing and mixing with the gun head or mixing upside down. For freeze-dried powder that is not easily dissolved, you can add buffer to it, and then place it on a horizontal shaker and shake it at low speed for a period of time; or put the Resuspending at 4°C for more than 2 hours to help it dissolve completely.

  1. Can PBS or DMEM be used to dissolve freeze-dried powder?

The dissolving buffer provided by the product is preferred. Different cytokines have different compositions of corresponding solutions. It is necessary to judge whether PBS or medium DMEM can be used according to the specific situation. In general, if the solution recommended by some freeze-dried powder is not PBS, it must not be dissolved by PBS, otherwise it will affect the activity of cytokines or recombinant proteins. f the recommended solution is 1XPBS, PBS can be used for dissolution.

  1. What substance is further used to dilute the solution containing the carrier protein? Is water ok?

Do not use water. The solution refers to a certain buffer capacity and neutral PH value, such as PBS, culture solution DMEM or RPMI1640.

 

  1. When doing serum-free culture or animal experiments, cytokines cannot contain BSA or HAS. What should we do if we want to preserve cytokines or recombinant proteins for a long time?

Order small packages, use one at a time, and run out in a short time.  Another method is to dilute the suspended cytokines or recombinant proteins with trehalose solution, and then pack and freeze them.

  1. How to calculate the active unit value of cytokines?

The calculation of activity unit value needs to be based on the median effective concentration and ED50 value of specific indicators. One ED50 is equivalent to one uint. For example, if the ED50 of a cytokine index is 2ng/ml, and the corresponding unit is 2ng/ml, then the active unit contained in 1mg of this index can be: units=1mg/(2.0ng/ml)=5*105

 

 

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