Sample Preparation with the Ultrasonic VialTweeter
Ultrasonic Sample Preparation
Ultrasonication is a common technique of sample treatment in order to get the sample ready for analyses such as polymers chain reaction (PCR), Western Blots, assays, molecular sequencing, chromatography etc. Ultrasonication is a technique widely used in laboratories to treat samples pre-analytically. A major advantage of sonication is that the working principle of ultrasound is based on purely mechanical forces. Ultrasonic lysis and cell disruption is achieved by sonomechanical shear forces, which gives ultrasonication the advantage that the solvents used for protein extraction can be used also during lysis. Ultrasonic cell disruptors such as the VialTweeter break the cell walls / membranes and promote mass transfer between cell interior and solvent. Thereby, the analyte (e.g. DNA, RNA, proteins, organelles etc.) are efficiently transferred from the cell matrix into the solvent. This means the steps of quenching and extraction overlap with the ultrasonic cell disruption process, which makes ultrasonic lysis very effective. Furthermore, ultrasonic sample preparation does not require detergents and other lysis reagents, which might alter and destruct the structure of the lysate and is known for the subsequent troubles with purification. Another lysis method, the enzymatic disruption requires long incubation times and delivers often non-reproducible results. Ultrasonic sample preparation overcomes common problems in sample preparation such as tissue homogenization, cell disruption, lysis, protein extraction and lysate solubilization. Since the intensity of the ultrasonic treatment can be exactly controlled and adjusted to the biological sample, degradation and sample loss are avoided. Automatically monitored and controlled sample temperature, pulse mode and sonication duration ensure optimum outcomes.
The VialTweeter is especially convenient for laboratory work that requires the simultaneous preparation of multiple samples under the same conditions. The VialTweeter is an ultrasonic block sonotrode that can hold up to 10 vials (e.g. Eppendorf, centriguge, NUNC tubes, cryo-vials) and sonicates them intensely under precisely controlled conditions. Since the ultrasonic energy is coupled through the walls of the vials into the sample medium, the vials maintain closed during the treatment. Thereby, sample loss and cross-contamination are completely avoided.
Vials and Tubes fitting the VialTweeter
The VialTweeter is suited to hold 10 common conical or round-bottom tubes such as Eppendorf, centrifuge, cryo-vials and various NUNC vial types, but the holes can be customized to other vial and tube sizes on request. Please let us know which kind of test tubes you want to use so that we can modify your VialTweeter accordingly. For larger test tubes such as Falcon tubes and other test containers, beakers and vessels, the VialPress is a convenient solution.
The VialTweeter with VialPress
Whilst the VialTweeter itself with its 10 tubes holes is already a unique and highly functional ultrasonic device, the VialPress add-on makes the VialTweeter even more versatile and flexible for operation. The VialPress is an accessory for the VialTweeter consisting in a clamp-on bar that allows to fixate larger sample tubes such as Falcon tubes or other small to mid-sized test beakers to the VialTweeter front. The picture on the left shows the VialTweeter holding 10 Eppendorf vials in the block, whilst the VialPress fixates one larger test tube to the front for sonication. The VialPress is capable to hold up to 5 larger test tubes for intense sonication.
VialTweeter Sample Preparation Protocols
The VialTweeter is widely used to sonicate biological samples. Before analysis, samples must be prepared for biochemical or biophysical analyses and assays, e.g. by lysis, tissue homogenization, protein extraction, DNA / RNA shearing, degassing etc. The VialTweeter fulfils these ultrasonic processes reliably and delivers reproducible results. A common application of the VialTweeter is the lysis/cell disruption of mammalian (human and animal) tissues as well as bacteria cells and viral particles. Successfully VialTweeter-treated biological samples include human lung epithelial cells, haematopoietic stem cells, myeloid leukemia cells, Escherichia coli, Bacillus subtilis, Bacillus anthracis, Francisella tularensis, Yersinia pestis, Streptococcus pyogenes, Caulobacter crescentus, Mycoplasma pneumoniae, mycobacteria / Mycobacterium tuberculosis complex (MTBC) and many other bacterial, botanical, and microbial cells.
Below, you can find a few selected protocols featuring the VialTweeter.
- tissue homogenization
- cell disruption & lysis
- protein extraction
- DNA / RNA shearing
- cell pellet solubilization
- pathogen detection
- in-vitro diagnostics
- pre-analytical treatment
E.Coli Lysis With VialTweeter for In-Vivo Glutathione Determination
Escherichia coli bacteria of the strain MG1655 were grown in MOPS minimal medium in a total volume of 200ml until an A600 of 0.5 was reached. The culture was split into 50-ml cultures for stress treatment. After 15 min of incubation with 0.79 mM allicin, 1 mM diamide, or dimethyl sulfoxide (control), cells were harvested at 4,000g at 4°C for 10 min. Cells were washed twice with KPE buffer prior to resuspension of pellets in 700µl of KPE buffer. For deproteination, 300µl of 10% (w/v) sulfosalicylic acid were added prior to disruption of cells by ultrasonication (3 x 1 min; VialTweeter ultrasonicator). Supernatants were collected after centrifugation (30 min, 13,000g, 4°C). Sulfosalicylic acid concentrations were decreased to 1% by the addition of 3 volumes of KPE buffer. Measurements of total glutathione and GSSG were performed as described above. Cellular glutathione concentrations were calculated based on a volume of E. coli cells of 6.7×10-15 liter and a cell density of A600 0.5 (equivalent to 1×108 cells ml-1 culture). GSH concentrations were calculated by subtraction of 2[GSSG] from total glutathione. (Müller et al. 2016)
Cell Lysis with VialTweeter before Graphite Furnace Atomic Absorption Spectrometry
Bacillus subtilis 168 (trpC2) were exposed to 15 min of antibiotic stress, then cells were harvested at 3,320 x g, washed five times with 100 mM Tris/1 mM EDTA, pH 7.5, resuspended in 10 mM Tris, pH 7.5 and disrupted by ultrasonication in a Hielscher VialTweeter instrument. (Wenzel et al. 2014)
VialTweeter Sample Preparation before Mass Spectrometry
The lyophilized cell pellets of human CD34 hematopoietic stem/progenitor cells were resuspended in 10µl (200µl for bulk HEK293 preparation for the peptide dilution series) of 8 M urea in 100 mM ammonium hydrogen carbonate and lysed aided by sonication with the Hielscher VialTweeter at an amplitude of 60%, a cycle of 60% and a duration of 20s for three times with intermediate cooling on ice. (Amon et al. 2019)
Sample Preparation Protocols using the VialPress
Fresh lettuce (Lactuca sativa) was homogenized in 0.5 M HEPES buffer (pH 8, KOH adjusted) at a ratio of 1 g plant (fresh weight) to 200, 100, 50, or 20 mL buffer solution. The ratio of plant mass to buffer solution volume was varied to keep the total homogenate volume between 3.5 and 12 mL. The ratio of plant mass to buffer solution volume was varied to keep the total homogenate volume between 3.5 and 12 mL, allowing homogenization with the probe. Homogenates then underwent indirect ultrasonication using an UP200St with VialTweeter equipped with 200xt VialPress (Hielscher Ultrasonics GmbH, Germany) for 3 min (80% pulse and 100% power). Using this device avoided contamination. (Laughton et al. 2019)
Reliable Temperature Control during Sonication with the VialTweeter
Temperature is a crucial process-influencing factor that is especially important for the treatment of biological samples. As all mechanical sample preparation techniques, sonication creates heat. However, the temperature of the samples can be well controlled when using the VialTweeter. We present you various options to monitor and control the temperature of your samples whilst preparing them with the VialTweeter and VialPress for analysis.
- Monitoring the sample temperature: The ultrasonic processor UP200St, which drives the VialTweeter, is equipped with an intelligent software and a pluggable temperature sensor. Plug the temperature sensor into the UP200St and insert the tip of the temperature sensor in one of the sample tubes. Via digital coloured touch-display, you can set in the menu of the UP200St a specific temperature range for your sample sonication. The ultrasonicator will automatically stop when the max temperature is reached and pause until the sample temperature is down to the lower value of the set temperature ∆. Then the sonication starts automatically again. This smart feature prevents heat-induced degradation.
- The VialTweeter block can be pre-cooled. Put the VialTweeter block (only the sonotrode without transducer!) into the fridge or freezer to pre-cool the titanium block helps to postpone temperature rise in the sample. If possible, the sample itself can be pre-cooled too.
- Use dry ice to cool during sonication. Use a shallow tray filled with dry ice and place the VialTweeter on the dry ice so that heat can rapidly dissipate.
Customers worldwide use the VialTweeter and VialPress for their daily sample preparation work in biological, biochemical, medical and clinical laboratories. The intelligent software and temperature control of the UP200St processor, temperature is reliably controlled and heat-induced sample degradation avoided. Ultrasonic sample preparation with the VialTweeter and VialPress delivers highly reliable and reproducible results!
Technical Details of the VialTweeter
The VialTweeter is a block sonotrode made from titanium that can hold up to 10 vials in the holes within the block. Additionally, up to 5 larger test tubes can be clamped to the VialTweeter front using the VialPress. The VialTweeter is so designed that the ultrasonication energy is distributed uniformly into each inserted vial to ensure reliable and uniform sonication results. A small pivot adjusts the VialTweeter sonotrode to uneven ground and align the test tubes vertically.
Advantages of the VialTweeter at a glance
- Intense sonication of up to 10 vials simultaneously
- Indirect sonication at high ultrasonic intensity through vessel wall into the sample
- Indirect sonication avoids cross-contamination and sample loss
- Reproducible results due to adjustable and controllable sonication amplitude
- The VialPress enables you to sonicate larger tubes
- Adjustable amplitude from 20 to 100%
- Adjustable pulse mode from 0 to 100%
The VialTweeter is powered by the UP200St, a 200 watts powerful ultrasonic processor. The UP200St is equipped with an intelligent software that allows for the precise control over all important ultrasonic process parameters such as amplitude, sonication time, pulsation, and temperature. This makes the VialTweeter a reliable tool for successful reproducible process results in biological and biochemical laboratories.
Amplitude can be adjusted between 20 and 100% and allows thereby to adapt the ultrasonic intensity to your sample. For instance, the shearing and fragmentation of DNA and RNA requires a milder amplitude to prevent the production of too small DNA fragments, the tissue homogenization of mouse brain needs high intensity sonication. Choose the ideal amplitude, sonication intensity and duration via the smart and intuitive menu at the UP200St processor. The menu and settings can be easily accessed and operated via coloured touch display. In the settings you can pre-set sonication parameters such as amplitude, pulsation / cycle mode, sonication duration, total energy input, and temperature limits. In research and production, the repeatability of trials and test results is crucial. This means the precise recording of process conditions and sonication protocols are utterly important. The automatic data protocolling writes all sonication data into a CSV file on an integrated SD-card so that you can easily check and compare various sonication runs. All ultrasonic process data can be easily accessed and shared as CSV file.
Hielscher Ultrasonics strives to provide you with advanced technology to facilitate and improve your research work!
Literature / References
- Müller A., Eller J., Albrecht F., Prochnow P., Kuhlmann K., Bandow J. E., Slusarenko A. J., Leichert L.I.O. (2016): Allicin Induces Thiol Stress in Bacteria through S-Allylmercapto Modification of Protein Cysteines. Journal of Biological Chemistry, Vol. 291, No. 22, 2016. 11477-11490.
- Tim Krischuns; Franziska Günl; Lea Henschel; Marco Binder; Joschka Willemsen; Sebastian Schloer; Ursula Rescher; Vanessa Gerlt; Gert Zimmer; Carolin Nordhoff; Stephan Ludwig; Linda Brunotte (2018): Phosphorylation of TRIM28 Enhances the Expression of IFN-β and Proinflammatory Cytokines During HPAIV Infection of Human Lung Epithelial Cells. Frontiers in immunology Vol. 9, September 2018.
- Michaela Wenzel, Alina Iulia Chiriac, Andreas Otto, Dagmar Zweytick, Caroline May, Catherine Schumacher, Ronald Gust, H. Bauke Albada, Maya Penkova, Ute Krämer, Ralf Erdmann, Nils Metzler-Nolte, Suzana K. Straus, Erhard Bremer, Dörte Becher, Heike Brötz-Oesterhelt, Hans-Georg Sahl, Julia E. Bandow (2014): Small Cationic Antimicrobial Peptides Delocalize Peripheral Membrane Proteins. PNAS April 8, 2014.
- Stephanie Laughton; Adam Laycock; Frank von der Kammer;Thilo Hofmann; Elizabeth A. Casman; Sónia M. Rodrigues; Gregory V. Lowry (2019): Persistence of copper-based nanoparticle-containing foliar sprays in Lactuca sativa (lettuce) characterized by spICP-MS. Journal of Nanoparticle Research 21:174, 2019.
- Sabine Amon, Fabienne Meier-Abt, Ludovic C. Gillet, Slavica Dimitrieva, Alexandre P. A. Theocharides, Markus G. Manz, Ruedi Aebersold (2019): Sensitive Quantitative Proteomics of Human Hematopoietic Stem and Progenitor Cells by Data-independent Acquisition Mass Spectrometry. Molecular & Cellular Proteomics 18, 2019. 1454–1467.