Ultrasonic Preparation of C. Elegans Samples
Ultrasonic Preparation of C. elegans Samples
C. elegans are roundworms, which are widely used in research laboratories to investigate genomics, developmental biology, and diseases. Many genes in the genome of C. elegans have functional counterparts in humans. Thereby, the nematode worm is an extremely useful model for human diseases. Other advantages for the wide use of C. elegans are its easy and cheap cultivation on plates containing bacteria (e.g., E. coli), its transparency, convenient handling, as well as the possibility to freeze and store the worms for a longer period.
Protein and lipid analysis are regular procedures in laboratories and ultrasonic sample preparation is the established method to lyse C. elegans nematodes in every developmental stage (i.e. embryos, larvae L1-L4, adults). Since C. elegans is also used as protein expression system to over-express targeted proteins, a reliable, reproducible lysis and protein extraction method, which gives high protein yields, is required. Ultrasonic cell disruption and extraction systems are available as probe-type homogenizers and as multi-sample ultrasonicators. Catering to convenient sample preparation and all kind of sample sizes numbers, Hielscher Ultrasonics has the ideal ultrasonic cell disruptor for your lab procedure.
- Preparation of worm homogenates
- Protein extraction
- Lipid extraction
- Protein quantification
- Western Blotting
- RNA extraction
- Enzymatic Assays
Ultrasonic Protocols for C. Elegans Disruption and Lysis
Ultrasonic homogenization and lysis of C. elegans and the subsequent protein and lipid extraction can be performed using varying procedures using different homogenization and lysis buffers etc. All lysis protocols have in common that the samples must be continuously kept on ice to prevent protein degradation. Below, we present you some reliable and rapid ultrasonic lysis and extraction protocols for the preparation of high-quality protein- or lipid-containing C. elegans samples.
Advantages of Ultrasonic C. elegans Lysis
- reliable process control
- gentle method
- easy to apply
Ultrasonic Protein Extraction from C. elegans Samples
Ultrasonic lysis and protein extraction of C. elegans worms can be performed using various protocols. Below we present you a few reliable and rapid lysis protocols for reproducible protein extraction results.
Rapid Preparatation of Cytosolic Extract from C. elegans Worms by Sonication
With the following protocol you can prepare C. elegans lysates in less than 30 minutes.
Collection of C. elegans
Pick the desired C. elegans worms into a 1.5ml tube phosphate buffered saline (PBS) or wash them from a plate with 1.5ml PBS. Centrifuge at for 1min at 2000rpm to pellet. Keep the samples at all time on ice.
Then, wash the worms twice with PBS.
Afterwards, wash the worms twice with ddH2O.
Resuspend the worms in at least 500ul of homogenization buffer (HB). The worm samples are now ready for ultrasonic lysis.
For higher protein extract quality, you might want to reduce bacterial contamination by washing the worms for 5min each in PBS and sterile, ultra-pure water (ddH2O) or perform sucrose floatation. Keep the worm samples continuously on ice.
Ultrasonic C. elegans Lysis Protocol
- Make sure that you prepare the ultrasonicator upfront so that the ultrasonic homogenizer is ready for use (probe mounted, sonication program pre-set).
- For C. elegans lysis with the UP200St or UP200Ht, ultrasonication should be performed using a microtip (e.g., 2mm probe S26d2; see picture left) at 40% amplitude for 1sec with 30sec pauses in-between. 5 sonication cycles for each 1 second with 30sec pauses are ideal for C. elegans lysis. If you perform the lysis for the first time, you can check the lysis progression in small aliquots of the sample after each pulse using a microscope.
- Lysis is completed successfully when the worms are disrupted. Over-sonication results in the breakage of nuclei and becomes visible when the sample gets viscous or foams. To prevent sample degradation, use more pulses if needed. Do not increase the time of each ultrasonic pulse cycle to get high-quality protein extracts.
- Clear cell lysate by centrifuging the ultrasonically lysed worms at 14,000rpm for 10min at 4ºC.
- Then, transfer the supernatant to a fresh tube and prepare for immunoprecipitation or other assays.
Note for homogenization buffer: Prepare the homogenization buffer for the ultrasonic lysis protocol above as follows:
- 15 mM Hepes pH 7.6 – 15 ml of 0.5 M
- 10 mM KCl – 2.5 ml of 2 M
- 1.5 mM MgCl2 – 0.75 ml of 1 M
- 0.1 mM EDTA – 100 ul of 0.5 M
- 0.5 mM EGTA – 2.5 ml of 0.1 M
- 44 mM Sucrose – 14.7 ml of 50 %
- Add right before use: 1mM DTT – 1000x of 1M
- plus a protease inhibitor
Ultrasonic Lysis of C. elegans for Quantitative Affinity Purification Assays
C. elegans embryos (∼2 million per replicate) were freshly harvested in biological triplicate by bleaching young gravid hermaphrodites and sonicated on ice (cycle: 0.5 s, amplitude: 40–45%, 5 strokes/session, 5 sessions, interval between sessions: 30 s; UP200S ultrasonic processor with micro-tip S26d2 (Hielscher Ultrasonics GmbH)) in lysis buffer (total volume: ∼600 μl; 50 mm Tris-HCl, pH 7.4, 100 mm KCl, 1 mm MgCl2, 1 mm EGTA, 1 mm DTT, 10% glycerol, protease inhibitor mixture, 0.1% Nonidet P-40 Substitute). After sonication, Nonidet P-40 Substitute was added up to 1% and the lysates were incubated with head over tail rotation at 4°C for 30 min, followed by centrifugation at 20,000 × g for 20 min at 4°C. Cleared lysate was then aspirated without disturbing the upper lipid layer and split by half into either the anti-GFP agarose beads or the blocked control beads (40–50 μl). After head over tail rotation at 4°C for 60–90 min, the beads were washed once with lysis buffer containing 0.1% Nonidet P-40 Substitute, followed by two times of washing in either buffer I (25 mm Tris-HCl, pH 7.4, 300 mm NaCl, 1 mm MgCl2) or buffer II (1 mm Tris-HCl, pH 7.4, 150 mm NaCl, 1 mm MgCl2) or both. For GFP:MBK-2 pull-downs, two separate experiments were performed using different washing conditions. Proteins were eluted by orbital shaking in 50 μl of 6 m urea/2 M thiourea at room temperature. For the MBK-1::GFP pull-down experiments, proteins were eluted twice by shaking in 50μl of 8 m guanidinium chloride at 90°C, followed by ethanol precipitation. Eluted protein samples were then digested in-solution.
(cf. Chen et al., 2016)
Ultrasonic Worm Homogenization and Lysis
For C.elegans lysis and protein extraction procedure, 30,000 nemotodes of the relevant stage were collected per sample and washed in ice-cold S-basal, concentrated by centrifugation at 1500 rpm for 2 min., washed six times with ice-cold S-basal to remove residual bacteria, and then stored on ice until ready for use. For protein extraction, gravid-adult worms were allowed to form a compact pellet after the last S-basal wash. Worm pellets were then resuspended in 1 ml of ice-cold Extraction Buffer [20 mM potassium phosphate, pH 7.4, 2mM EDTA, 1% Triton-X-100, protease inhibitors (Sigma P2714)] and processed immediately.
∼30,000 gravid-adult worms (corresponding to ∼100 mg wet weight), were sonicated on ice using a a probe-type ultrasonicator (e.g. UP50H with microtip MS2) at 40% amplitude for 10 cycles of 3 sec on, 30 sec off, in 1 ml of ice-cold extraction buffer. (cf. Baskharan et al. 2012)
Ultrasonic Lipid Extraction from C. elegans
In lipidomics, a branch of metabolomics, the lipid complement of biological systems is characterized and analysed. C. elegans are widely used in lipidomics to investigate the interaction of metabolic lipids and their effects on health and lifespan.
Ultrasonic lysis and extraction is used to release lipids such as sphingolipids from C. elegans embryos, larvae, and adult worms. Ultrasonication is used to prepare worm homogenates and subsequently to extract the lipids from the sample.
Protocol for Ultrasonic Lipid Extraction from C. elegans
Thaw the C. elegans pellet on ice and resuspend with 0.5 ml ultrawater. Sonicate the C. elegans samples in 1,5mL test tubes, whilst keeping the samples continuously on ice.
Sonication can be performed using a probe-ultrasonicstor such as the UP200Ht, the ultrasonic sample prep unit VialTweeter (simultaneous sonication of 10 samples) or the UIP400MTP (for the sonication of multi-well plates such as 96-well plates). For ultrasonic lysis with the UP200Ht use the micro-tip S26d2. Pre-set the ultrasonic cycle mode in the digital menu. Set amplitude to 10% and sonication cycle mode of 2 sec pulses of 20 cycles with a pause of 30 sec. between each ultrasonic pulsation burst.
Transfer the supernatants to glass tubes with screw cap. Perform Folch extraction by adding 1 ml ultrawater to each glass tube, followed by adding 6 ml of chloroform/methanol (ratio = 2:1) mixture to each glass tube.
Vortex each glass tube for 30 sec for 4 times. Centrifuge the tubes at 1,258 x g for 15 min (Eppendorf, 5810 R) to further enhance phase separation. Transfer the lower hydrophobic fraction to a clean glass tube by a glass Pasteur pipette. Dry the lower hydrophobic fraction under nitrogen flow in a nitrogen evaporator. Store the dried pellet in a -80 °C freezer until use.
Ultrasonic Preparation of Worm Lysates
Worm lysate: L4 stage worms were harvested and washed three times with M9 buffer (42.26 mM Na2HPO4, 22.04 mM KH2PO4, 85.56 mM NaCl, and 0.87 mM MgSO4) in order to remove all bacteria. After removing as much as possible of M9 buffer, worms were resuspended in lysis buffer: 50 mM HEPES, 50 mM KCl, 1mM EDTA, 1mM EGTA, 5 mM phosphate β-glycerol, 0.1% (v/v) Triton X-100, 50 mM sodium fluoride, 1 mM sodium orthovanadate, 5 mM sodium pyrophosphate, 0.2 mM phenylmethanesulfonylfluoride and protease inhibitor. Worms were frozen in liquid nitrogen and thawed at 37°C for three times, then worms were sonicated on dry ice with an ultrasonic VialTweeter unit for the simultaneous preparation of 10 sample tubes. Sonication was carried out at 50% amplitude in 10 cycles of 2 sec. with 30 sec pause between the sonication bursts. Afterwards, the samples were centrifuged at 12000 rpm at 4°C for 15 min. The supernatant was collected and stored at -70°C. An aliquot was used for protein quantification by Bradford assay.
Determination of total glutathione, GSH, and GSSG: For glutathione quantification, lysates and determination were made the same day. Glucose-fed and control L4 larvae were harvested and washed with M9 buffer three times. After removing as much as possible of M9 buffer, worms were resuspend in ice-cold metaphosphoric acid (5% w/v), then worms were sonicated in ice with an ultrasonic VialTweeter at 50% amplitude in ten sonication cycles of 2 sec. with 30 sec. pause between each cycle. Afterwards, centrifuged at 12000 rpm at 4 ̊C for 15 min.
(cf. Alcántar-Fernández et al., 2018)
C. elegans Sample Prep before Immunoprecipitation and Western Blotting
In brief, for embryonic extracts, C. elegans L1 larvae were grown in large-scale liquid S-medium cultures to adulthood. Embryos were collected using the standard bleach method and suspended in lysis buffer (50 mM Tris, pH 7.5, 100 mM KCl, 1 mM EDTA, 1 mM MgCl2, 8.7% glycerol, 0.05% NP-40, 1 Protease Inhibitor Cocktail, and 1 Phosphatase Inhibitor Cocktail I and II), quickly frozen in liquid nitrogen, and lysed by ultrasonic cell disruption using an ultrasonicator with microtip such as the UP200Ht with S26d2 for 10 pulses over 10 s at 30% amplitude. If a larger number of samples must be prepared the ultrasonic VialTweeter or the MultiSample-Ultrasonicator UIP400MTP for well-plates are recommended. After sonication, extracts were pre-cleared by centrifugation at 30,000g for 20 min at 4°C. The pre-cleared extract (300µg of total protein) was incubated with 40µg of anti-CDC-25.1 affinity-purified antibody (this study) cross-linked to protein A-agarose, or as control, a similar amount of rabbit immunoglobulin (Ig)G cross-linked to protein A-agarose was used in a total volume of 200µl of lysis buffer containing 1% NP-40, the inclusion of which reduced nonspecific binding of proteins to the matrix. The samples were rotated for 1 h at 4°C, the beads were washed three times with lysis buffer, and eluted with 30µl of glycine/HCl and 200 mM NaCl, pH 2.2. After immunoprecipitation, eluates were diluted in 30µl of SDS sample buffer, heated to 95°C for 4 min, and typically 3% of total for input and 30% for the eluates were applied to SDS-PAGE followed by Western blotting with anti-CDC-25.1 (1:400), anti-LIN-23 (1:750), anti-ubiquitin (1:1000), anti-GSK3 (1:500), or anti-β-actin (1:2000) antibodies. Where extracts were not subjected to immunoprecipitation, the same amount of total protein derived from those extracts was resuspended in SDS sample buffer, heated to 95°C, and then directly applied to SDS-PAGE and analyzed by Western blotting. (cf. Segref et al. 2020)
Ultrasonic Lysis under Prescise Temperature Control
The precise and reliable temperature control is crucial when handling biological samples. High temperatures initiate thermally-induced protein degradation in 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.
Find the Optimal Ultrasonic Cell Disruptor for your Lysis Application
Hielscher Ultrasonics is long-time experienced manufacturer of high-performance ultrasonic cell disrupters and homogenizers for laboratories, bench-top and industrial scale systems. Your bacterial cell culture size, your research or production goal and the volume of cell to process per hour or day are essential factors to find the right ultrasonic cell disruptor for your application.
Hielscher Ultrasonics offers various solutions for the simultaneous sonication of multi-samples (up to 10 vials) as well as mass samples (i.e., microtiter plates / 96-well plates), the classic probe-type lab ultrasonicator with different power levels from 50 to 400 watts to fully industrial ultrasonic processors with up to 16,000watts per unit for commercial cell disruption and protein extraction in large production. All Hielscher ultrasonicators are built for the 24/7/365 operation under full load. Robustness and reliability are core features of our ultrasonic devices.
All digital ultrasonic homogenizers are equipped with smart software, coloured touch display and automatic data protocolling, which make the ultrasonic device into a convenient work tool in lab and production facilities.
Let us know, what kind of cells, what volume, with what frequency and with what target you have to process your biological samples. We will recommend you the most suitable ultrasonic cell disruptor for your process requirements.
The table below gives you an indication of the approximate processing capacity of our ultrasonic systems from compact hand-held homogenizers and MultiSample Ultrasonicators to industrial ultrasonic processors for commercial applications:
|Batch Volume||Flow Rate||Recommended Devices|
|96-well / microtiter plates||n.a.||UIP400MTP|
|10 vials à 0.5 to 1.5mL||n.a.||VialTweeter at UP200St|
|0.01 to 250mL||5 to 100mL/min||UP50H|
|0.01 to 500mL||10 to 200mL/min||UP100H|
|10 to 2000mL||20 to 400mL/min||UP200Ht, UP400St|
|0.1 to 20L||0.2 to 4L/min||UIP2000hdT|
|10 to 100L||2 to 10L/min||UIP4000hdT|
|n.a.||10 to 100L/min||UIP16000|
|n.a.||larger||cluster of UIP16000|
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Literature / References
- Chen J.-X; Cipriani P.G.; Mecenas D.; Polanowska J.; Piano F.; Gunsalus K.C.; Selbach M. (2016): In Vivo Interaction Proteomics in Caenorhabditis elegans Embryos Provides New Insights into P Granule Dynamics. Molecular & Cellular Proteomics 15.5; 2016. 1642-1657.
- Jonathan Alcántar-Fernández, Rosa E. Navarro, Ana María Salazar-Martínez, Martha Elva Pérez-Andrade, Juan Miranda-Ríos (2018): Caenorhabditis elegans respond to high-glucose diets through a network of stress-responsive transcription factors. PLoS One 13(7); 2018.
- Segref, A.; Cabello, J.; Clucas, C.; Schnabel, R.; Johnstone I.L. (2010): Fate Specification and Tissue-specific Cell Cycle Control of the Caenorhabditis elegans Intestine. Molecular Biology of the Cell Vol. 21, 2010. 725–738.
- Henderson S.T., Bonafe M., Johnson T.E. (2006): daf-16 protects the nematode Caenorhabditis elegans during food deprivation. J Gerontol A Biol Sci Med Sci. 2006; 61:444–60.
Facts Worth Knowing
C. elegans is a free-living transparent nematode (roundworm) about 1 mm in length, which feeds on bacteria (e.g. E. coli) and has a relatively short life cycle. At 20 °C, the laboratory strain of C. elegans (N2) has an average lifespan around 2–3 weeks and a generation time of 3 to 4 days. When C. elegans are grown in large numbers, which can be easily done under precisely controlled lab conditions, they can be easily screened for the working principle of novel drugs as well as their effects and interaction within complex molecular processes in human disease. The short genome, short life cycle and simple handling in laboratory settings make C. elegans an ideal model organism for research such as genomics, proteomics, developmental biology, disease research, drug development etc.
Caenorhabditis elegans worms can be either male or hermaphrodite. Hermaphrodites have both male and female reproductive organs. However, female worms do not exist. Hermaphrodites can either self-fertilise or can also breed with male worms. C. elegans can produce over 1,000 eggs every day.
Since C. elegans is one of the simplest organisms with a nervous system, the nematode worm is used since 1963 as a model organism for research. The neurons do not fire action potentials, and do not express any voltage-gated sodium channels. In the hermaphrodite, this system comprises 302 neuron the pattern of which has been comprehensively mapped, in what is known as a connectome.
Many of the genes in the C. elegans genome have functional counterparts in humans which makes it an extremely useful model for human diseases and is for instance used to study developmental biology, ageing and factors influencing longevity. Furthermore, C. elegans mutants provide models for many human diseases including neurological disorders (e.g. Alzheimer’s), congenital heart disease and kidney disease.
These factors made C. elegans a highly valuable model for many research fields. Consequently, C. elegans was the first multicellular organism to have its whole genome sequenced. The genome contains an estimated 20,470 protein-coding genes. About 35% of C. elegans genes have human homologs. Remarkably, human genes have been shown repeatedly to replace their C. elegans homologs when introduced into C. elegans. Conversely, many C. elegans genes can function similarly to mammalian genes.
The lifespan of C. elegans is approx. 3 weeks and consists of six life stages: embryogenesis (egg stage), four larval stages (L1 to L4), and the adult stage. The nematodes hatch from eggs as L1 larvae comprised of 560 cells. Growth during each larval stage occurs by cell division and by cell hypertrophy. Cuticular molting punctuates each larval stage. If harsh environmental conditions signal to the developing worm that conditions are unlikely to support adult fertility, C. elegans can alter its development and form an alternate L3 larval stage, where the larvae go into a dauer stage. In this state, animals are extraordinarily stress-tolerant and long-lived and can survive three to nine months. Dauer larvae isolate themselves from adversity by sealing both their buccal and anal cavities, shrinking their gut, and turning on a daf-16/FOXO-dependent genetic program that, among other things, leads to expression of a dauer-specific cuticle. (cf. Henderson et al., 2006)
C. elegans Dauer Larvae
Dauer larvae is the term for nematode larvae, which entered an alternative developmental stage. ther term “Dauer larvae” is particularly used for worms of the rhabditids family including Caenorhabditis elegans. The word “Dauer” is of German origin and means the “duration” in the meaning of “a period of time”. Dauer larvae go into a type of stasis and can survive harsh conditions. If and when a larva enters the dauer stage is dependent on environmental conditions. Dauer larvae are extensively studied in biology because the laravae show an extraordinary ability to survive harsh environments and live for extended periods of time. For example, C. elegans dauer larvae can survive up to four months, much longer than their average lifespan of about three weeks during normal reproductive development.