Sonication for Cell Lysis: Cell Disruption and Extraction
Ultrasonic cell lysis is a sample preparation procedure in biotech laboratories. The goal is to disrupt cell walls or entire cells to release biological molecules. Sonication is commonly used for cell lysis, cell disruption and extraction. The major advantage of sonicators for cell lysis lies in the precise control of process parameters, such as intensity and temperature, allowing for gentle yet efficient cell disruption and extraction.
Cell Lysis using Ultrasound
Ultrasonic cell lysis uses high-frequency sound waves to break open cells and extract their contents. Sonication is established and reliable for cell disruption and extraction of intracellular material, such as plasmids, receptor assays, proteins, DNA, and RNA. By adjusting process parameters, the ultrasonic intensity can be finely tuned to meet specific application requirements, ranging from gentle to intense sonication. The steps following lysis include fractionation, organelle isolation, and protein extraction and purification. The resulting lysate must be separated for further investigations or applications, such as proteomic research.
If you would like to learn more about sonication for lysis, cell disruption and extraction, please contact us. Our technical team will be glad to work with you on your cell lysis project.
Ultrasonic homogenizers UP100H (100 watts) and UP400St (400 watts): Sonication for Cell lysis and Extraction.
Advantages of Using Sonication for Cell Lysis
Compared to other cell lysis and extraction methods, ultrasonic cell lysis has several advantages:
- Tezlik: Ultrasonic cell lysis and extraction is a fast method that can break open cells in a matter of seconds. This is much faster than other methods such as homogenization, freeze-thawing or bead-milling.
- Samaradorlik: Ultrasonic cell lysis and extraction can be used to treat small, large or multiple samples at once, making it more efficient than other methods that require individual processing of small samples.
- Chemical-free: Ultrasonic cell lysis and extraction is a non-invasive method that does not require the use of harsh chemicals or enzymes. This makes it ideal for applications where the integrity of the cell contents needs to be maintained. Unwanted contamination of samples can be avoided.
- High yield: Ultrasonic cell lysis and extraction can extract a high yield of cellular contents, including DNA, RNA, and proteins. This is because the high-frequency sound waves break open the cell walls and release the contents into the surrounding solution.
- Harorat nazorati: Sophisticated ultrasonciators allow for precise temperature control of the sample. Hielscher digital sonicators are equipped with a pluggable temperature sensor and temperature monitoring software.
- Reproducible: Protocols for ultrasonic cell lysis can be easily reproduced and even matched to different larger or smaller sample volumes by a simple linear scale-up.
- Versatile: Ultrasonic cell lysis and extraction can be used to extract a wide range of cell types, including bacteria, yeast, fungi, plant and mammalian cells. It can also be used to extract different types of molecules, including proteins, DNA, RNA, and lipids.
- Simultaneous preparation of numerous samples: Hielscher Ultrasonics offers several solutions to comfortably process numerous samples under the exactly same process conditions. This makes the sample preparation step of lysis and extraction highly efficient and time-saving.
- Easy to use: Ultrasonic cell lysis and extraction equipment is easy to use and requires minimal training. The equipment is also economical as it is a single investment with no requirement for re-purchasing of disposals. This makes it attractive for a wide range of researchers and laboratories.
Overall, ultrasonic cell lysis and extraction is a fast, efficient, precisely controllable, and versatile method for extracting cellular contents. Its advantages over alternative methods make it an attractive choice for a wide range of research and industrial applications.
Working Principle of Ultrasonic Cell Lysis
Ultrasonic cell lysis and extraction uses high-frequency sound waves to disrupt cells and extract their contents. The sound waves create pressure changes in the surrounding liquid, causing small bubbles to form and collapse in a process known as cavitation. These bubbles generate localized highly intense mechanical forces that can break open cells and release their contents into the surrounding solution.
Cell lysis using an ultrasonicator commonly involves the following steps:
- The sample is placed in a tube or container with a liquid buffer.
- An ultrasonic probe is inserted into the sample, and high-frequency sound waves with approx. 20-30 kHz are applied.
- The ultrasound waves cause oscillation and cavitation in the surrounding liquid, generating localized forces that break open cells and release their contents.
- The sample is centrifuged or filtered to remove any cellular debris, and the extracted contents are collected for downstream analysis.
Powerful ultrasound waves disrupt the cell matrix of biological structures and release the bioactive compounds. Mass transfer between the plant material and the solvent (e.g., buffer) is intensified. (graphic: ©Vilkhu et al., 2006)
Disadvantages of Common Lysis Methods
During your work in laboratories, you might have already experienced the hassle of cell lysis using traditional mechanical or chemical lysis protocols.
- Mechanical lysis: Mechanical lysis methods, such as grinding with mortar and pestle or homogenization using a french press, bead mill, or rotor-stator system often lack precice control and adjustment options. This means the use of milling and grinding can quickly generate heat and shear forces that can damage the sample and denature proteins. They can also be time-consuming and require large amounts of starting material.
- Chemical lysis: Chemical lysis methods, such as detergent-based lysis, can damage the sample by disrupting the lipid bilayer and denaturing proteins. They can also require multiple steps and may leave residual contaminants that interfere with downstream applications. Finding the optimum dosage of detergent is an additional challenge.
- Freeze-thaw cycles: Freeze-thaw cycles can cause cell membranes to rupture, but repeated cycles can also cause protein denaturation and degradation. This method can also require multiple cycles, which can be time-consuming and often results in lower yields.
- Enzymatic lysis: Enzymatic lysis methods can be specific to certain cell types and require multiple steps, making them time-consuming. They also generate waste and require careful optimization to avoid degradation of the sample. Enzymatic lysis kits are often expensive. If your current enzymatic lysis procedure gives insufficient results, sonication as synergistic method can be applied to intensify cell disruption.
In contrast to conventional mechanical and chemicals cell lysis methods, sonication is a very efficient and reliable tool for cell disintegration that allows for a complete control over the sonication parameters. This ensures a high selectivity on materials release and product purity. [cf. Balasundaram et al., 2009]
It is suitable to all cell types and easily applicable in small and large scale – always under controlled conditions. Ultrasonicators are easy to clean. An ultrasonic homogenizer always features clean-in-place (CIP) and sterilize-in-place (SIP) function. The sonotrode consists in a massive titanium horn which can be wiped or flushed in water or solvent (depending on the working medium). The maintenance of ultrasonicators is due to their robustness almost neglectable.
Ultrasonik liziz va hujayra buzilishi
Generally, the lysis of samples in the lab will take between 15 seconds and 2 minutes. As the intensity of sonication is very easy to adjust by amplitude setting an sonication time as well as by choosing the right equipment, it is possible to disrupt cell membranes very gently or very abruptly, depending on the cell structure and on the purpose of lysis (e.g. DNA extraction requires softer sonication, complete protein extraction of bacteria requires a more intense ultrasound treatment). The temperature during the process can be monitored by an integrated temperature sensor and can be easily controlled by cooling (ice bath or flow cells with cooling jackets) or by sonication in pulsed mode. During pulse-mode sonication, short sonication burst cycles of 1-15 seconds duration allow for heat dissipation and cooling during the longer intermittent periods.
All ultrasound-driven processes are completely reproducible and linearly scalable.
The VialTweeter is an ultrasonic homogenizer for simultaneous, uniform and rapid sterile preparation of numerous samples.
Ultrasonic Homogenizers for Cell Lysis and Extraction
Various types of ultrasonic devices allow for matching the sample preparation goal and to assure user-friendliness and operation comfort. Probe-type ultrasonicators are the most common devices in the lab. They are most suitable for the preparation of small and mid-size samples with volumes of 0.1mL up to 1000mL. Different power sizes and sonotrodes allow to adapt the ultrasonicator to the sample volume and the vessel for most effective and efficient sonicating results. The ultrasonic probe device is the best choice when single samples have to be prepared.
If more samples have to be prepared, e.g. 8-10 vials of cell solution, an intense indirect sonication with ultrasonic systems such as the VialTweeter or an ultrasonic cuphorn are the most suitable homogenization method for an efficient lysis. Several vials are sonicated at the same time, at the same intensity. This saves not only time, but ensures also the same treatment of all samples, which makes the results among the samples reliable and comparable. Furthermore, during indirect sonication cross-contamination by immersing the ultrasonic sonotrode (also known as ultrasonic probe, horn, tip, or finger) is avoided. Since individually to sample size matched vials are used, time-consuming clean-up and sample loss due to decanting of vessels are omitted. For the uniform sonication of multiwell or microtiter plates, Hielscher offers the UIP400MTP.
For higher volumes, e.g. for the commercial production of cell extracts, continuous ultrasonic systems with a flow cell reactor are most suitable. The continuous and even flow of the processed material assures an even sonication. All parameters of the ultrasonic disintegration process can be optimized and adjusted to the requirements of the application and the specific cell material.
Exemplary procedure for ultrasonic lysing of bacterial cells:
- Preparation of the cell suspension: Cell pellets must be completely suspended in a buffer solution by homogenizing (choose your buffer solution compatible with following analysis, e.g. specific chromatography method). Add lysozymes and/ or other additives, if needed (they must be also compatible with separation/ purification means). Mix/ homogenize the solution gently under mild sonication until complete suspension is achieved.
- Ultrasonic lysis: Place the sample in an ice bath. For cell disruption, sonicate the suspension at 60-90 second bursts (using pulse mode of your sonicator).
- Separation: Centrifuge the lysate (e.g. 10 min. at 10,000 x g; at 4degC). Separate the supernatant from the cell pellet carefully. The supernatant is the total cell lysate. After filtration of the supernatant, you obtain a clarified fluid of the soluble cell protein.
The most common applications for ultrasonicators in biology and biotechnology are:
- Cell extract preparation
- Disruption of yeast, bacteria, plant cells, soft or hard cell tissue, nucleic material
- protein ekstraktsiyasi
- Preparation and isolation of enzymes
- Production of antigens
- DNA extraction and/ or targeted fragmentation
- lipozoma tayyorlash
Cell disruption with probe-type ultrasonicator is an efficient sample preparation method.
The table below gives you an overview over our ultrasonicators for cell disruption and extraction. Click at the device type to get more information on each ultrasonic homogenizer. Our well-trained and long-time experiences technical staff will be glad to help you choosing the most suitable ultrasonicator for your samples!
| To'plam hajmi | Oqim darajasi | Tavsiya etilgan qurilmalar |
|---|---|---|
| up to 10 vials or tubes | na | VialTweeter |
| multiwell / microtiter plates | na | UIP400MTP |
| multiple tubes / vessels | na | chashka |
| 1 dan 500 ml gacha | 10 dan 200 ml / min | UP100H |
| 10 dan 1000 ml gacha | 20-200 ml/min | UP200Ht, UP200St |
| 10 dan 2000 ml gacha | 20 dan 400 ml / min | UP400St |
The manifold applications of ultrasound branches out in the sectors of biotechnology, bioengineering, microbiology, molecular biology, biochemistry, immunology, bacteriology, virology, proteomics, genetics, physiology, cellular biology, hematology, and botany.
Lysis: Breaking Cell Structures
Cells are protected by a semi-permeable plasma membrane which consists in a phospho-lipid bilayer (also protein-lipid bilayer; formed by hydrophobic lipids and hydrophilic phosphorus molecules with embedded protein molecules) and creates a barrier between the cell interiors (cytoplasm) and the extracellular environment. Plant cells and prokaryotic cells are surrounded by a cell wall. Due to multiple layers thick cell wall of cellulose, plant cells are harder to lyse than animal cells. The cell interior, such as organelles, nucleus, mitochondrion, is stabilized by the cytoskeleton.
By lysing the cells, it is aimed at extracting and separating the organelles, proteins, DNA, mRNA or other biomolecules.
Conventional Methods of Cell Lysis and their Drawbacks
There are several methods to lysate cells, which can be divided into mechanical and chemical methods, which include the use of detergents or solvents, the application of high pressure, or the use of a bead mill or of a french press. The most problematic disadvantage of these methods is the difficult control and adjustment of process parameters and thereby impact.
The table below displays main disadvantages of common lysis methods:
Table: Conventional methods of cell lysis have major disadvantages
Procedure of Lysis
Lysis is a sensitive process. During the lysis the protection of cell membrane is destroyed, however the inactivation, denaturation and degradation of the extracted proteins by an unphysiological environment (deviation from pH-value) must be prevented. Therefore, in general lysis is carried out in a buffer solution. Most difficulties arise from uncontrolled cell disruption resulting in an untargeted release of all intracellular material or/ and the denaturation of the target product.
Frequently Asked Questions about Sonication and Cell Lysis
- Can you lyse cells with sonication? Yes, sonication effectively lyses cells using high-frequency ultrasonic waves that induce cavitation, a phenomenon where tiny vapor bubbles form and collapse violently within the cell suspension. The resulting mechanical forces disrupt cell membranes and facilitate the release of intracellular components into the liquid.
- How to use a sonicator for cell lysis? Utilizing a sonicator for cell lysis involves immersing the sonicator probe into a cell suspension and adjusting parameters such as amplitude and pulse duration. The process should be closely monitored to optimize cell disruption while minimizing protein denaturation and enzyme inactivation.
- What is the principle of sonication for cell lysis? Sonication operates on the principle of acoustic cavitation. Ultrasonic energy is transmitted into the liquid medium, causing rapid pressure fluctuations that lead to the formation and implosion of microbubbles. These implosions generate intense shear forces and localized high temperatures, disrupting cellular structures and enhancing lysate homogeneity.
- How long does cell lysis sonication take? The duration of sonication for cell lysis can vary significantly depending on factors like cell type, cell density, sonicator power, and the specific protocol used. Typical procedures may range from several seconds to a few minutes, often performed in cycles to manage heat generation and ensure uniform cell disruption.
- What is the purpose of sonication in protein extraction? In protein extraction, sonication serves to efficiently rupture cell membranes and solubilize proteins. This method is particularly useful for releasing proteins from within cellular compartments, making it essential for preparing lysates from which proteins are to be purified or analyzed.
- Why is sonication used for extraction? Sonication is favored for extraction due to its rapid action and ability to apply targeted energy, breaking down cellular structures to release bioactive molecules without the use of harsh chemical treatments, thereby preserving the functional integrity of the extracted compounds.
- Does sonication disrupt protein-protein interactions? While sonication can effectively disrupt cell membranes, it may also disrupt protein-protein interactions. The level of disruption depends on the sonication intensity and exposure duration, potentially leading to denaturation or dissociation of protein complexes, which might affect subsequent analytical or functional studies.
- Can sonication be used to lyse E. coli? Hielscher sonicators are particularly effective for lysing bacterial cells such as E. coli, which have robust cell walls. The technique provides a physical method to shear the cell wall and membrane, making it a preferred method for preparing bacterial lysates in molecular biology and biochemistry labs.
Adabiyot/Adabiyotlar
- Balasundaram, B.; Harrison, S.; Bracewell, D. G. (2009): Advances in product release strategies and impact on bioprocess design. Trends in Biotechnology 27/8, 2009. pp. 477-485.
- Vilkhu, K.; Manasseh, R.; Mawson, R.; Ashokkumar, M. (2011): Ultrasonic Recovery and Modification of Food ingredients. In: Feng/ Barbosa-Cánovas/ Weiss (2011): Ultrasound Technologies for Food and Bioprocessing. New York: Springer, 2011. pp. 345-368.
- Nico Böhmer, Andreas Dautel, Thomas Eisele, Lutz Fischer (2012): Recombinant expression, purification and characterisation of the native glutamate racemase from Lactobacillus plantarum NC8. Protein Expr Purif. 2013 Mar;88(1):54-60.
- Brandy Verhalen, Stefan Ernst, Michael Börsch, Stephan Wilkens (2012): Dynamic Ligand-induced Conformational Rearrangements in P-glycoprotein as Probed by Fluorescence Resonance Energy Transfer Spectroscopy. J Biol Chem. 2012 Jan 6;287(2): 1112-27.
- Claudia Lindemann, Nataliya Lupilova, Alexandra Müller, Bettina Warscheid, Helmut E. Meyer, Katja Kuhlmann, Martin Eisenacher, Lars I. Leichert (2013): Redox Proteomics Uncovers Peroxynitrite-Sensitive Proteins that Help Escherichia coli to Overcome Nitrosative Stress. J Biol Chem. 2013 Jul 5; 288(27): 19698–19714.
- Elahe Motevaseli, Mahdieh Shirzad, Seyed Mohammad Akrami, Azam-Sadat Mousavi, Akbar Mirsalehian, Mohammad Hossein Modarressi (2013): Normal and tumour cervical cells respond differently to vaginal lactobacilli, independent of pH and lactate. ed Microbiol. 2013 Jul; 62(Pt 7):1065-1072.