How To Choose The Appropriate Ultrafiltration Tube
Ultrafiltration tubes commonly used for protein concentration and buffer replacement are often used in ultrafiltration tubes. There are other models, which have different volume sizes and ultrafiltration tubes, depending on the molecular weight of the target protein, the volume before concentration and the target volume of concentration. Reusable.
1. Select a suitable ultrafiltration tube, mainly considering MWCO and concentration volume. Generally, the molecular weight should not be greater than 1 / 3 of the molecular weight of the target protein. If the molecular weight of the target protein is about 10 kd, an ultrafiltration tube with a retained molecular weight of 3 KD can be used.
2. The newly purchased ultrafiltration is dry. Add milliq water before use. The water passes completely through the membrane and is precooled in an ice bath or refrigerator for a few minutes. Then pour out the water to add the protein solution. The amount of protein solution shall not exceed the white line on the top of the test tube. Easy operation. Before adding the protein solution, the ultrafiltration tube needs to be inserted into the ice for pre cooling.
3. Balance. The mass and center of gravity must be balanced. Note that the speed and acceleration should not be too fast, otherwise the ultrafiltration membrane will be directly damaged. Start centrifugal ultrafiltration (precooling the centrifuge to 4 degrees). The direction of the diaphragm and the rotating shaft can be adjusted according to the instructions (in the case of angle centrifuge, the diaphragm is perpendicular to the shaft). In actual use, the general speed is lower than that specified in the manual, which may prolong the service life of the centrifugal pipe.
4. when concentrated to the remaining 1ml, [take the 50ul household Bradford solution, add 10ul fluid to see if it turns blue, and determine if the ultrafiltration tube leaks the protein. If the tube leaks, pour it into the upper layer and then flow back to the new tube to start ultrafiltration. To accurately determine whether the test tube leaks, please 5mg 10min centrifuge 10min, then pass it through, run the protein gel or Bradford] [coarse measurement]. Continue to add the remaining protein solution to concentrate (operate on ice to prevent protein heating) until all concentrated solutions are added. During centrifugation, pay attention to whether protein precipitation occurs and causes tube blockage. If precipitation occurs, please determine the specific cause of precipitation, whether the protein concentration is too high or whether the buffer is inappropriate; The former can be solved by using multiple ultrafiltration tubes at the same time to reduce the concentration, while the latter can change different buffers until the protein precipitates.
5. The previous steps are used to concentrate protein. If the buffer is to be replaced, when the total protein solution is concentrated to about 1ml, gently add a new buffer (ultrafiltration through 0.22um ultrafiltration membrane), and then continuously concentrate to about 1ml for three times, so that the final concentration depends on the required protein concentration, which is usually not more than 500ul or less than 200ul. According to the volume concentration of at least 10 times each time, it is more than 1000 times that of three times, which can basically achieve the purpose of changing the buffer.
6. Remove the final protein concentrate. Operate on ice. Using a yellow pipette tip (200ul), gently insert the pipette tip along the edge, then gently blow and mix the protein solution. Be careful not to touch the ultrafiltration membrane. Suck the concentrated solution to 200ul each time until it is completely sucked. It is not necessary to extract the last concentrated solution at the bottom of the test tube, otherwise it is too difficult and the ultrafiltration membrane may be damaged. Finally, milliq water is added to the ultrafiltration tube without passing through the ultrafiltration membrane to prevent water loss and drying of the membrane.
7. The following are the steps for treating the ultrafiltration tube and reusing the ultrafiltration tube.
8. Pour water into the ultrafiltration tube and gently rinse with milliq water for several times. [if there is obvious protein precipitation at the bottom of the test tube, you can add water first and then blow it away with a pipette. Be careful not to touch the membrane. The sediment hangs down and then runs away. You can't flush it with tap water. After adding 0.2m NaOH solution, place it at room temperature for 20 minutes, and balance the ultrafiltration tube during this period. Centrifuge for another 10 minutes. Pour out the remaining NaOH solution and immerse the cotton core in the beaker (1 or 2L) Milliq water, placed for several hours, and then replaced with new water, placed for several hours to continuously dilute the concentration of NaOH. Wash the 50ml test tube and cover with tap water, and wash the inner wall with milliq water.
9. Take out the immersed test tube core, add it to the full milliq water, and then fill the 50ml test tube with milliq water. Slowly put the tube core into 50ml.
Centrifuge tube, drain and divide water, then cover and store at 4 degrees until next use. Generally speaking, according to the above steps and precautions, each tube will not break within three or four years.
