Density gradient centrifugation method, also known as zone centrifugation.

The method'sadvantageIt can simultaneously separate several or all components in the sample, with good resolution and separation effect, and can obtain relatively pure components at once.

shortcomingThe centrifugation time is relatively long, requiring the preparation of gradient liquid and high operational requirements.

According to the principle of centrifugal separation, density gradient centrifugation can be further divided intoRate zone centrifugation method（rate zonal centrifugation method，R-Z） 和Equal density centrifugation method (isopycnic centrifugation method)。

Rate zone centrifugation, also known as equal zone density centrifugation, is the centrifugal separation and purification of mixed samples based on the different volume sizes and sedimentation coefficients of different component particles in the sample.

During centrifugation, the sample solution to be separated is placed on a gradient liquid column formed by density gradient materials such as sucrose, cesium chloride (CsCI), *, etc. The density of the sample particles must be greater than the density at any point in the gradient liquid column, otherwise the ideal zone centrifugation effect cannot be obtained. During centrifugation, different components in the mixed sample will form their own zones at different positions of the gradient liquid column. Suitable rotation speed and time are selected for centrifugation. When the distance between each zone is extended, centrifugation is terminated and the zones are removed.In this way, each component in the mixed sample can be separated and purified by only one centrifugation, and its purity and recovery rate can meet the requirements.

advantageThis method is suitable for separating and purifying samples with a sedimentation coefficient difference of more than 20%. It has high resolution and skilled operation, and can effectively separate samples with a sedimentation coefficient difference of 5% to 10%.

shortcomingDue to material limitations, the concentration of the sample solution cannot be too high, otherwise the operating conditions are difficult to control. Additionally, compared to differential centrifugation, this method requires much less sample processing.

Figure 1 Speed zone with centrifugation

The centrifugation time in the rate zone must be strictly controlled and cannot be too short, otherwise the sample zone will be unclear and the time cannot be too long, otherwise the components to be separated may sink to the bottom. The settling velocity of the sample particles that need to be separated depends on factors such as particle shape, size, density, and centrifugal force. The state of the sample before and after centrifugation is shown in Figure 1.

The classic application of rate zone centrifugation is to purify various subcellular organelles (ribosomes, mitochondria, exosomes, etc.) and viral particles (viral vectors, vaccines, etc.) through sucrose density gradient centrifugation. For example, the 70S ribosome precipitate we obtained in the previous phase can be further separated and purified using sucrose density gradient centrifugation after hydrolysis to obtain two subunits, 50S and 30S. The specific experimental process is as follows:

Centrifugal Classroom is being serialized and we will continue to follow and learn more about centrifugal methods!

Pay attention to previous centrifuge classes

□Common centrifugal terms - centrifugal force and relative centrifugal force

□Settlement coefficient of commonly used terms

□Preparation of exosome free serum by rapid centrifugation method

□Rotation efficiency K factor and centrifugation time

□Differential centrifugation | Did you choose the right separation and purification method?

□The sterilization troubles in the laboratory can be solved by Xiaobei Mall at a 50% discount!

*Approval number: RA-051-cent-adv-ruo

*Revision date: October 16, 2020

*Disclaimer: No unauthorized modifications are allowed. Beckman Coulter reserves the right to update versions at any time without prior notice