Concentrate method: not only can use a plastic straw to […]
Concentrate method: not only can use a plastic straw to draw and collect immediately, but also put the centrifuge tube into the centrifuge to back-spin, throw the concentrated liquid into the collection cap, cover and store. Both of these two methods can make the concentrated liquid almost completely collected;
There are 3k, 5K, 10K, 30K, 50K, 100k, 300K, 1000K and μ l PES membranes in the system;
The newly released patented technology, hydroart membrane 2K, has the characteristics of very low protein adsorption, high water flow and high-throughput sequencing. It is an ideal membrane for immunoglobulin concentration and desalination.
(1) Select suitable ultrafiltration tube. Generally, the molecular weight of the target protein should not exceed 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 a molecular weight of 10kDa can be selected. If the molecular weight of the target protein is above or below 10kd, the ultrafiltration tube with molecular weight of 5kd can be used. Read the manual carefully and pay attention to the different tolerance levels of ultrafiltration membrane to various compounds.
(2) The newly bought ultrafiltration is dry. Add ultra pure water system before application, and the water flow is thoroughly through the membrane. Cool in ice bath or refrigerator for several minutes. Then pour in the water, you can add the protein solution, how much is added, with no more than a line at the top of the tube as the standard. The actual operation should be light. Before adding protein solution, the ultrafiltration tube must be inserted on the ice surface for rapid cooling.
(3) Equilibrium. Quality and focus must be balanced. Pay attention to the speed ratio and instantaneous speed, or the ultrafiltration membrane will be destroyed immediately. At the beginning of ultrafiltration (centrifuge quenched to 4 degrees). The rotational speed ratio of different centrifuges is different when rpm is calculated as G. The instantaneous speed of the centrifuge is adjusted to the minimum gear to reduce the working pressure on the membrane. Note that you must wait for the centrifuge to achieve the target speed ratio, then you can leave the centrifuge. Otherwise, when the centrifuge has a problem, you can't solve it at the first time. The orientation of the film and the transmission shaft is adjusted according to the instructions (the condition of the angular centrifuge is that the film and the shaft are vertical). In the specific application, the general speed is lower than that in the operating instructions, which can increase the service life of the centrifugal tube.
(4) When concentrated to the remaining 1ml, take 50ul buffer solution, add 10ul flow through to see if it turns dark blue, so as to distinguish whether the ultrafiltration tube jumps off the protein. If the tube leaks, pour the top layer and flow through into the new tube again and start ultrafiltration. In order to accurately distinguish whether the tube is leaking or not, use 5mgml BSA to filter for 10min, then take the flow through, run through the protein glue or Bradford rough measurement, add the remaining protein solution again to concentrate (in the actual operation on ice surface, avoid the protein from getting hot), until all the concentrated solution is full. In the whole process of filtration, pay attention to whether protein sedimentation occurs, causing blockage of the pipeline. If sedimentation occurs, it is necessary to determine whether the actual reason for sedimentation is that the protein concentration value is too high or the buffer is not suitable; the former method can use multiple ultrafiltration tubes to reduce the concentration value, while the latter method is to change different buffer solutions until the protein does not produce sedimentation.
(5) The first two steps are used to concentrate the protein. If the buffer has to be changed, when the total protein solution is concentrated to about one milliliter, a new buffer is added gently (ultrafiltration by 2 um ultrafiltration membrane), and then concentrated to about one milliliter for three times. The final volume of the last concentration is determined according to the required protein concentration value. Generally, it does not exceed 500 UL, and it is also concentrated to within 200 UL.