Cell Disruption: Sonication vs. Homogenization

样品的制备蛋白质和核酸acid analysis requires two essential steps: disruption of the tissue to release individual cells and lysis of those cells to release their cellular contents. Mechanical homogenization and sonication (sometimes referred to as ultrasonic homogenization) are two mainstay techniques for these processes.

为您的特定应用程序选择适当的技术应该仔细考虑。每个人都有其好处和缺点，实验室经常选择相互使用这些技术。继续阅读以获取信息，以帮助选择一种避免破坏珍贵或不可替代的组织样品的方法。

Mechanical Homogenization

Mechanical homogenization utilizes direct physical force to bring a biological sample in solution to a state of uniform distribution, such that all fractions’ molecular composition is consistent. Traditionally, mechanical disruption was achieved by freezing tissues and then grinding with a mortar and pestle. Presently, there are two other common methods: bead-based disruption and rotor-stator disruption. These separation techniques efficiently homogenize samples, but often require further downstream fractionation to obtain the desired concentration or purity of molecules.

基于珠的均质化使用塑料或金属珠和高速摇动来产生剪切力。该技术非常适合整个动物，昆虫（例如，Drosophila melanogaster）或植物标本，需要破坏坚固的细胞壁。基于珠的破坏的缺点是它需要正确选择珠材料和直径。例如，某些塑料珠将很容易与DNA结合，在去除时将其从溶液中耗尽。同样，珠子的直径会影响产生的力量和样品中断的程度。使用太大或太小的珠子大小会导致不完整的均匀化。值得庆幸的是，许多仪器制造商都开发了各种珠子，以适合大多数均质化应用。

Rotor-stator disruption involves the use of a stationary stator housing a rapidly moving rotor. The movement of tissues and cells within the space between the rotor and stator creates a high degree of shearing force. These devices are typically hand-held and the rotor-stator attachment is interchangeable. These devices efficiently disrupt tough animal and plant tissues (e.g., muscle). One salient benefit of rotor-stator disruption is the ability to progressively increase the degree of homogenization by sequentially processing with increasingly smaller rotor-stator distance attachments. This process can effectively homogenize even the most resilient specimens. Additionally, very large-volume samples can be processed using larger disruption instrumentation. Drawbacks of rotor-stator disruption include increased cost and more cumbersome equipment management. Attachments often require washing and sterilization prior to reuse. Disposable attachments are offered, but with a higher operating cost.

Ultrasonic Homogenization

Ultrasonic homogenization also utilizes mechanical forces to shear tissues and cells, however, this force takes the form of ultrasonic sound waves. This can be done either using an ultrasonic bath or a probe. This technique can be used to rapidly homogenize samples, but care must be given to the power and frequency used, as well as the duration of the processing, since improper sonication parameters can result in irreversible sample damage.

Both bath and probe sonicators utilize high-energy sound waves to disrupt samples. Probe sonication is the most frequently used process for the disruption of cells. Bath sonication is useful for large batch preparations of tissues and cells.

从整体组织开始时，请使用组织类型来确定单独的超声处理是否是有效破坏的可行技术。更多的弹性组织，例如肌肉，需要上游的主要破坏，例如混合，快速冻结或机械均质化。还值得一提的是，整个动物和某些植物组织无法单独通过超声处理来完成。因此，尽管超声处理有效，快速破坏溶液和许多组织中的细胞，但不能用于某些起始材料。

Ultrasonic homogenization is a high-energy process and operators run the risk of altering the molecular make-up of the solution during sonication. Users must specify the frequency and wattage of the process. Nucleic acids, in particular, are susceptible to the shearing forces of sonication. If a sample is processed at too high of a frequency, the DNA/RNA backbone is sheared and analysis is no longer feasible. The susceptibility of nucleic acids to shearing can also benefit operators. If your desired end product is protein, users will want to remove the DNA in solution. Sonication can effectively disrupt genomic and cellular nucleic acids, so that they no longer interfere with protein purification. Lastly, the high-energy created through sonication can heat samples and result in the denaturation of proteins (if the sample is processed for an extended duration). Thus, be mindful of the duration, wattage and frequency used for ultrasonic homogenization.

The homogenization of tissues and cells is a common technique used to facilitate further downstream analysis. Myriad techniques and instruments are offered for these processes. Mechanical homogenization and ultrasonic homogenization are two commonly used techniques. Each has its advantages and drawbacks. Many times researchers chose to use the two techniques in concert in order to obtain the most desirable outcome. A summary of how they compare:

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