Precautions for use of ultrafiltration tube
(1) Select the appropriate ultrafiltration tube. Generally, the molecular weight to be retained should not be greater than 1 / 3 of the molecular weight of the target protein. For example, if the molecular weight of the target protein is 35kDa, the ultrafiltration tube with 10kDa molecular weight can be selected. If the molecular weight of the target protein is about 10kd, an ultrafiltration tube with a molecular weight of 3KD can be used. Carefully read the instruction manual and pay attention to the different tolerance of ultrafiltration membrane to various chemicals.
(2) The newly bought ultrafiltration is dry. Add ultrapure water before use, and the water will completely pass through the membrane. Pre cool it in an ice bath or refrigerator for a few minutes. Then pour out the water and add the protein solution. The amount added shall not exceed the white line on the top of the tube. The operation should be light. Before adding protein solution, the ultrafiltration tube needs to be inserted on ice for precooling.
(3) Balance. Both mass and center of gravity should be balanced. Note that the speed and acceleration should not be too fast, otherwise the ultrafiltration membrane will be directly damaged. Start centrifugal ultrafiltration (centrifuge precooling to 4 degrees). After the RPM of different centrifuges is converted into G, it is different. Adjust the acceleration of the centrifuge to the lowest gear to reduce the pressure on the membrane. Note that you must wait until the centrifuge reaches the target speed before leaving the centrifuge, otherwise when there is a problem with the centrifuge, you can't deal with it at the first time. The direction of the membrane and the rotating shaft shall be adjusted according to the instructions (in the case of angle rotating centrifuge, the membrane is perpendicular to the shaft). In practical use, the speed is generally lower than that in the manual, which can prolong the service life of the centrifugal pipe.
(4) When it is concentrated to the remaining 1ml, take 50ul buffer solution and add 10ul to flow through to see if it turns blue, so as to judge whether the ultrafiltration tube leaks protein. If the tube leaks, pour the upper layer and flow through the new tube and start ultrafiltration. To accurately judge whether the tube is leaking, centrifuge with 5mgml BSA for 10min, then take flow through, run protein glue or Bradford for rough measurement, and continue to add the remaining protein solution for concentration (operate on ice to prevent the protein from being heated) until all the concentrated solutions are added. Pay attention to whether protein precipitation occurs during centrifugation, resulting in pipe blockage. If precipitation occurs, determine the specific cause of precipitation, whether the protein concentration is too high or the buffer is inappropriate; The former can be solved by simultaneous ultrafiltration with multiple ultrafiltration tubes to reduce the concentration. The latter is to change different buffer solutions until the protein does not precipitate.
(5) The previous steps are used to concentrate protein. If you want to change the buffer, when the total protein solution is concentrated to about 1ml, gently add a new buffer (ultrafiltration through 0.22um ultrafiltration membrane), and then concentrate to about 1ml for three consecutive times. The final volume of the last concentration depends on the required protein concentration, which is generally not more than 500ul, or it can be concentrated to less than 200ul. According to the volume concentration of at least 10 times each time, it can reach more than 1000 times three times, which can basically achieve the purpose of changing the buffer.
