Either synchronous adult worms (~200,000) or early embryos (from 10 plates of 100,000 adults) were collected, washed three times with M9 buffer, suspended in lysis buffer composed of 20 mM HEPES (pH 7.5), 125 mM sodium citrate, 0.1% (v/v) Tween 20, 0.5% (v/v) Triton X-100, 2 mM MgCl2, 1 mM DTT, and a Mini Protease Inhibitor Cocktail Tablet (Roche), and homogenized in a FastPrep-24 benchtop homogenizer (MP Biomedicals). Worm or embryo lysates were centrifuged twice at 14,000 × g for 30 min at 4°C, and supernatants were incubated with GBP beads for 1.5 hr at 4°C on a rotating shaker. The beads were washed three times with IP buffer containing protease inhibitor for 5 min each wash, and then washed twice with high-salt wash buffer (50 mM HEPES pH 7.5, 500 mM KCl, 0.05% NP40, 0.5 mM DTT, and protease inhibitor). Immune complexes were eluted with elution buffer (50 mM Tris-HCl pH 8.0, 1× SDS) for 10 min at 95°C.
Fastprep 24 benchtop homogenizer
Manufactured by MP Biomedicals
Sourced in United States
The FastPrep-24 is a benchtop homogenizer designed for efficient sample preparation. It uses high-speed mechanical disruption to break down a variety of sample types, including tissues, cells, and microorganisms, into a homogeneous suspension. The device operates at adjustable speeds to accommodate different sample requirements.
Automatically generated - may contain errors
Lab products found in correlation
Mini protease inhibitor cocktail tablet, by Roche (1 mentions)
Turbovap evaporator, by Biotage (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti rabbit hrp conjugated secondary antibody, by GE Healthcare (1 mentions)
Genegnome device, by Syngene (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Nanodrop, by Thermo Fisher Scientific (1 mentions)
Centrivap, by Labconco (1 mentions)
Fastprep lysing matrix b tubes, by MP Biomedicals (1 mentions)
7 protocols using fastprep 24 benchtop homogenizer
1
Worm Protein Extraction and Immunoprecipitation
2
Immunoprecipitation of Protein Complexes
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Synchronous adult worms were collected and washed three times with M9 buffer. The worms were then homogenized in a FastPrep-24 benchtop homogenizer (MP Biomedicals). Worm extracts were centrifuged at 14000 × g for 15 mins at 4 °C. The worm extracts were incubated with anti-FLAG or GFP trap beads for 2 hrs at 4 °C on a rotating shaker. The beads were washed three times and subjected to SDS-PAGE. Signals were detected using corresponding primary and secondary antibodies.
3
Metabolite Extraction from Cell Pellets
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Chilled Lysing Matrix C beads (MP Biomedicals) were added to frozen cell pellets on dry ice. Chilled methanol was then added to the tube containing beads and pellets. Samples were disrupted on FastPrep-24™ Benchtop Homogenizer (MP Biomedicals) and centrifuged at maximum speed for 10 min. Supernatant was transferred into an Eppendorf® Protein LoBind Tube and dried using a TurboVap® Evaporator (Biotage). The evaporated samples were reconstituted in methanol, vortexed briefly, centrifuged, and the supernatant was collected in an amber screw-top vial (Waters). The metabolite extract was analyzed immediately on LC–MS or stored temporarily at −20 °C prior to analysis.
4
Bacterial Cell Lysis and Lysate Preparation
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Bacterial cells from overnight cultures standardized as described above were harvested by centrifugation and resuspended in 750 μl of ice-cold TEG buffer (25 mM Tris at a pH of 8 and 25 mM EGTA). Cell suspensions were then transferred to 2 ml RNase/DNase free Fastprep Lysing Matrix B tubes (MP Biomedicals, cat. # 116911050). Cell suspensions were then lysed in a FastPrep®-24 benchtop homogenizer (MP Biomedicals, Solon, OH) for two separate 40 second intervals at a rate of 6.0 m/sec (interrupted by a 5 minute interval in which the homogenates were chilled on ice). After centrifugation at 15,000 X g at 4oC for 10 minutes, supernatants were aliquoted and stored at -20oC until use.
5
Analyzing Sup35 and Rnq1 Proteins in Yeast
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Strains were grown to midlog phase either in YPD or in YNB glucose. Protein extraction was performed according to the published protocol (Kushnirov et al., 2006 (link)) but with a modified lysis buffer (100 mM Tris-HCl, pH 7.5, 50 mM NaCl, 10 mM 2-mercaptoethanol, 2% [vol/vol] protease inhibitor cocktail [Sigma, USA], 2 mM phenylmethylsulfonyl fluoride [PMSF]). Cells were homogenized with a FastPrep-24 benchtop homogenizer (MP Biomedicals, USA) at 6.0 M/S for 30 s and then incubated on ice for at least 30 s. The procedure was repeated seven times. The SDD-AGE was performed according to the published protocol (Kryndushkin et al., 2003 (link); Drozdova et al., 2020 (link)). Gels with 1.5% (wt/vol) agarose were run at 30 V for 200–240 min. Proteins were transferred onto a polyvinylidene fluoride membrane according to the published protocol (Halfmann and Lindquist, 2008 (link)). The rabbit polyclonal anti-Sup35 (SE4290, Chabelskaya et al., 2004 (link)) and anti-Rnq1 (unpublished data) antibody were used to detect Sup35 or Rnq1, respectively. The HRP–conjugated secondary anti-rabbit antibody (GE Healthcare) was used for detection. Signals were recorded with the GeneGnome device (SynGene).
6
DNA Isolation from Plant and Microbes
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DNA from plant and microbial samples was isolated using the FastDNA Spin Kit for Soil (MP Biomedicals LLC, Solon, United States) according to the manufacturer’s instructions. For the cell lysis, a FastPrep-24 benchtop homogenizer (MP Biomedicals) was used for 2 × 60 s at 6 m/s with a 5 min cooling period on ice between runs. To obtain fresh cell material for isolation from pure cultures, both fungi and bacteria were grown on their respective growth media with a sterilized layer of cellophane or filter paper placed between agar surface and microbe. The cell material was then carefully scraped off using one-way sterile stainless steel blades (Feather Safety Razor Co., Ltd., Osaka, Japan). The material was either immediately used for isolation or first frozen in liquid nitrogen and subsequently lyophilized using a benchtop vacuum centrifuge (CentriVap; Labconco Corporation, Kansas City, MO, United States). The isolated DNA was eluted in 100 μl of DNase and pyrogen-free water before being stored at −20°C in aliquots of 50 μl or at 4°C between experiments. The DNA concentration and the absorbance ratio at 260/280 nm were determined with Nanodrop (2000/2000c; Thermo Fisher Scientific, Waltham, MA, United States).
7
Bacterial Growth Phase Proteome Profiling
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Whole-cell lysates were prepared as previously described with minor modification (45 (link)). Briefly, strains were cultured at 37°C in TSB with constant shaking and a medium-to-flask ratio of 0.5. Bacterial cells from a volume of each culture calculated to obtain an equivalent number of cells were harvested by centrifugation at an OD560 of approximately 1.5, 4.0, and 10.0, which correspond to the mid‐exponential, late‐exponential, and post‐exponential growth phases, respectively. Cells were washed with sterile phosphate-buffered saline (PBS) and resuspended in 750 μl of TEG buffer (25 mM Tris‐HCl [pH 8.0], 25 mM EGTA). Cell suspensions were stored at −20°C until all samples had been collected, at which point samples were thawed on ice, transferred to Fastprep Lysing Matrix B tubes, and lysed in a FastPrep-24 benchtop homogenizer (MP Biomedicals) using two 40-s intervals at a rate of 6.0 m/s interrupted by a 5-min interval during which the homogenates were chilled on ice. After centrifugation at 15,000 × g for 10 min at 4°C, supernatants were harvested and stored at −80°C.
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