Say No to Contamination and Denaturation! Key Summary of Centrifuge Separation Techniques

　　From relevant literature, it is understood that... Centrifuge The centrifugal separation method continues to hold advantages such as its broad separation range and large capacity. It allows for the study of the hydrodynamic properties of native biomacromolecules, enables fragment separation of biomacromolecules in ionizing media, and imposes minimal restrictions on buffer selection (in contrast to electrophoresis, where the thermal effects of current severely limit the choice of buffers). Moreover, centrifugation remains widely employed during the pretreatment stages of various experimental procedures. Considering the distribution of equipment across different research departments, centrifugal separation still enjoys considerable prevalence under many conditions. As an effective method for separation and purification, differential centrifugation, rate-zonal density gradient centrifugation, and isopycnic centrifugation continue to play a crucial role in the study of biomacromolecules.

　　Key points for centrifuge separation:

　　1) Prevent pollution:

　　To avoid degradation or denaturation of biomacromolecules during experiments due to partial damage, it’s important to identify the causes of degradation. There are many factors that can lead to degradation, such as external forces applied during the experimental process and contamination by certain enzymes. However, the most common cause is enzymatic activity.

　　During the experiment, contamination caused certain dehydrogenases to enter the sample, leading to the denaturation of biomacromolecules.

　　(2) Other precautions to avoid denaturation:

　　(i) Before centrifugation, pre-cool the rotor to a temperature close to the intended centrifugation temperature (either by overnight refrigeration or by maintaining it in a temperature-controlled incubator). For ultracentrifuges, the cooling system is primarily designed to counteract the heat generated by aerodynamic friction as the rotor spins at high speeds in thin air—specifically, as the linear velocity of the rotor surface increases from subsonic to supersonic levels. In a vacuum, where only radiative heat transfer occurs, it takes a long time—typically around one hour—for the temperature of a rapidly spinning rotor to drop from room temperature (e.g., 30–35°C in summer) to 5°C. Therefore, it is generally advisable to pre-cool the rotor to a temperature close to the intended centrifugation temperature (±2°C) to ensure that, during the initial phase of centrifugation (within the first 15 minutes), the rotor temperature and the sample temperature remain consistent.

　　(ii) When performing gradient centrifugation using a fixed-angle rotor, a near-vertical tube rotor, or a vertical tube rotor, it is essential to utilize the centrifuge’s slow acceleration function (0–500 rpm) and slow deceleration function (500 rpm–0) to ensure a smooth transition of the gradient direction.

　　(iii) At the start and end of centrifugation, handle and remove the centrifuge tubes carefully and gently to avoid large vibrations.

　　(iv) The sample-unloading process should be carried out on a vibration-free workbench under conditions where the ambient temperature is similar to the centrifugation temperature. The sample-unloading method should be gentle and involve minimal external forces; whenever possible, samples should not be conveyed using pumps.

　　3) Gradient centrifugation also has its own key points. This topic is relatively complex and requires consulting numerous references. Friends who are interested can explore it on their own.

　　(1) B.D. Hames, “Choice of Conditions for Density Gradient Centrifugation,” IRL Press, Oxford, 1984

　　(2) M. Dobrota, R. Hinton, “Conditions for Density Gradient Separations,” Oxford University Press, 1992

　　(3) D. RickWood, B.D. Hames, “Centrifugation, Essential Data,” Wiley Press, 1994

　　(4) C.A. Price, “Centrifugation in Density Gradients,” Academic Press, New York, 1982

　　(5) Yu Xingming, “Ultracentrifugal Separation of Plasmid DNA,” Chemistry of Life, 1994.1

　　(6) Yu Xingming, “Centrifugal Separation of Serum Lipoproteins,” Chemistry of Life, March 1997

　　(7) Yu Xingming, “Introduction to Centrifugal Technology” Equipment and Methods, Shanghai Society of Biophysics, 1986, 1993.

　　The above is a brief introduction to centrifuges. Although a centrifuge is just a small-scale separation device, its functions and value are significant. Therefore, the more you learn about centrifuges, the more you’ll be able to uncover their full potential and value!