0.5 mL ultrafiltration centrifuge tube

Avoid excessively high rotational speeds or acceleration, as they can directly damage the ultrafiltration membrane. Before starting, pre-chill the centrifuge to 4 °C. Note that RPM corresponds to relative centrifugal force ($g$) differently across centrifuge models. Always set the acceleration and deceleration to the lowest rate to minimize pressure on the membrane. In practice, running the centrifuge at a speed slightly below the recommended speed in the manual helps extend the tube's service life.

Monitor the process closely to ensure protein precipitation does not occur and clog the filter. If precipitation occurs, determine the underlying cause—whether it is due to excessively high protein concentration or an unsuitable buffer system: 1. High Concentration: Divide the sample across multiple ultrafiltration tubes to lower the localized protein concentration during centrifugation. 2. Unsuitable Buffer: Systematically test and switch to a different buffer until the protein remains stable without precipitating.

1. Follow standard concentration steps until the sample volume is reduced to approximately 1 mL. 2. Gently add the new buffer and concentrate back down to ~1 mL. 3. Repeat this process 3 times in total. Assuming a minimum 10-fold volume concentration per cycle, three rounds achieve an overall exchange ratio exceeding 1,000-fold, effectively completing the buffer exchange. 4. For the final concentration step, reduce the volume according to your desired final protein concentration—typically to no more than 500 $\mu\text{L}$, or even below 200 $\mu\text{L}$ for higher concentrations.

1. Perform all recovery steps on ice using a 200 ul pipette tip. 2. Carefully insert the tip along the inner wall of the filter insert and gently pipette up and down to mix the protein solution—avoid touching or scratching the ultrafiltration membrane. 3. Aspirate the concentrated solution in increments close to 200 $\mu\text{L}$ until fully recovered. Do not attempt to recover the tiny residual amount at the absolute bottom, as doing so risks puncturing the membrane. 4. Immediately add ultrapure water to submerge the membrane and prevent it from drying out.

1. Discard the water, gently rinse the filter insert several times with ultrapure water, and add 0.1 M NaOH solution. 2. Allow it to stand at room temperature for 20 minutes, then centrifuge for 10 minutes and pour out the residual NaOH solution. 3. Submerge the filter insert in a beaker containing 1–2 L of ultrapure water for several hours. Replace with fresh ultrapure water and soak for another few hours to thoroughly dilute and wash away residual NaOH. 4. Wash the outer tube (e.g., 50 mL tube) and cap with tap water, followed by a thorough rinse of the inner walls with ultrapure water. 5. Fill the submerged filter insert nearly to the top with ultrapure water, and fill the outer tube as well. Gently place the insert back into the outer tube (allowing excess water to overflow), screw the cap on tightly, and store at 4 °C until next use.