Usage and precautions of ultrafiltration centrifuge tube
1) Choose a suitable ultrafiltration tube. Note that UF membranes differ in their level of tolerance to various chemicals. Typically, ultrafiltration tubes with a 10 kDa molecular weight cut-off can be selected with a molecular weight cut-off that should not be greater than 1 / 3 of the molecular weight of the protein of interest, such as 35 kDa. If the molecular weight of the protein of interest is around 10 kd, a ultrafiltration tube with a molecular weight cut-off of 3 KD can be used.
(2) The ultrafiltration, freshly bought, is dry, with ultrapure water added before use and water completely passed through the membrane, ice bath or refrigerator pre cooled for a few minutes. The water is then poured off, i.e. the protein liquid, and how much is added, whichever is not more than the white line at the top of the tube. The operation is light, and the ultrafiltration tube needs to be precooled on ice before adding the protein solution.
3) Both the mass and center of gravity were to reach the balance. Note the rotational speed and acceleration are not very fast, otherwise directly damaging the ultrafiltration membrane. Centrifugal ultrafiltration was started (centrifuge precooled to 4 degrees). After the RPM of different centrifuges was converted to g, it was different. The acceleration of the centrifuge was adjusted to a minimum, reducing the pressure on the membrane. Note, be sure to leave the centrifuge after the centrifuge has reached its destination speed, or if you have a problem, you can’t handle it for the first time. The orientation of the membrane to the spindle was adjusted according to the instructions (angular centrifuge case is membrane to axis perpendicular). In practical use, the general rotation speed is lower than that in the instructions, so that the life of the centrifuge tube can be extended.
3) Both the mass and center of gravity were to reach the balance. Note the rotational speed and acceleration are not very fast, otherwise directly damaging the ultrafiltration membrane. Centrifugal ultrafiltration was started (centrifuge precooled to 4 degrees). After the RPM of different centrifuges was converted to g, it was different. The acceleration of the centrifuge was adjusted to a minimum, reducing the pressure on the membrane. Note, be sure to leave the centrifuge after the centrifuge has reached its destination speed, or if you have a problem, you can’t handle it for the first time. The orientation of the membrane to the spindle was adjusted according to the instructions (angular centrifuge case is membrane to axis perpendicular). In practical use, the general rotation speed is lower than that in the instructions, so that the life of the centrifuge tube can be extended.
(4) When concentrated to the remaining 1ml, take 50ul of buffer solution, add 10ul of flow through and see if there is any blue colour, as a judgement whether the ultrafiltration tube is missing protein. If the tube is leaky, re pour the upper layer and flowthrough into a new tube to start ultrafiltration. To determine precisely if tubes were missed, centrifuge for 10min with 5mgml of BSA prior to flowthrough, running on protein glue, or Bradford crude assay, and continue by adding the remaining concentrated protein solution (which operates on ice and prevents proteins from heating) until all of the concentrate has been added. Take care during centrifugation if precipitation of proteins occurs, leading to tube closure. If precipitation occurs, determine whether the specific cause of precipitation is an excessive protein concentration or an inappropriate buffer; The former can be solved by ultrafiltration of multiple ultrafiltration tubes simultaneously, decreasing the concentration, and the latter by exchanging different buffer solutions until no precipitation of the protein occurs.
(4) When concentrated to the remaining 1ml, take 50ul of buffer solution, add 10ul of flow through and see if there is any blue colour, as a judgement whether the ultrafiltration tube is missing protein. If the tube is leaky, re pour the upper layer and flowthrough into a new tube to start ultrafiltration. To determine precisely if tubes were missed, centrifuge for 10min with 5mgml of BSA prior to flowthrough, running on protein glue, or Bradford crude assay, and continue by adding the remaining concentrated protein solution (which operates on ice and prevents proteins from heating) until all of the concentrate has been added. Take care during centrifugation if precipitation of proteins occurs, leading to tube closure. If precipitation occurs, determine whether the specific cause of precipitation is an excessive protein concentration or an inappropriate buffer; The former can be solved by ultrafiltration of multiple ultrafiltration tubes simultaneously, decreasing the concentration, and the latter by exchanging different buffer solutions until no precipitation of the protein occurs.
(5) The first few steps are used to concentrate the protein, and if the buffer is to be changed, gently add new buffer (ultrafiltration through a 0.22um ultrafiltration membrane) to about 1ml of total protein, and reconcentrate to about 1ml for three consecutive times, with the final concentrated final volume depending on the desired protein concentration, generally not more than 500ul, but also to within 200ul. Achieve 1000 × or more on three occasions, essentially as much buffer change, as calculated by at least 10 × or so volume enrichment each time.
(5) The first few steps are used to concentrate the protein, and if the buffer is to be changed, gently add new buffer (ultrafiltration through a 0.22um ultrafiltration membrane) to about 1ml of total protein, and reconcentrate to about 1ml for three consecutive times, with the final concentrated final volume depending on the desired protein concentration, generally not more than 500ul, but also to within 200ul. Achieve 1000 × or more on three occasions, essentially as much buffer change, as calculated by at least 10 × or so volume enrichment each time.
Post time: Nov-09-2022