Water bath sonicator

Manufactured by Diagenode

Sourced in United States

The Water bath sonicator is a laboratory equipment piece designed to generate ultrasonic waves in a water bath. Its core function is to provide a controlled environment for sonication processes, which can be used for various applications such as sample preparation, cell lysis, and more.

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Lab products found in correlation

11 protocols using water bath sonicator

Proteomics Workflow for Skeletal Muscle and Aorta

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Tibialis anterior and descending thoracic aorta were collected as described above, individually pulverized in liquid nitrogen, and resuspended in about 5 volumes of LYSE buffer (PreOmics), heated at 95 °C for 5 min under continuous shaking and sonicated in a water-bath sonicator (Diagenode) for 15 min with a 50% duty cycle. Protein concentration in the lysate was measured with a fluorometric method based on a tryptophan standard curve [29 (link)] and subsequently adjusted to 2 μg/μl. Proteolytic digestion was carried out by adding 1 μg of endoproteinase LysC and trypsin per 50 μg of lysate. After overnight digestion at 37 °C under continuous shaking, the lysate was acidified to a final concentration of 0.1% trifluoro-acetic acid (TFA) and loaded onto StageTip plugs of SDB-RPS. Purified peptides were eluted with 80% acetonitrile - 1% ammonia and dried. Tibialis anterior samples were analyzed in technical triplicates.

Ducommun S., Jannig P.R., Cervenka I., Murgia M., Mittenbühler M.J., Chernogubova E., Dias J.M., Jude B., Correia J.C., Van Vranken J.G., Ocana-Santero G., Porsmyr-Palmertz M., McCann Haworth S., Martínez-Redondo V., Liu Z., Carlström M., Mann M., Lanner J.T., Teixeira A.I., Maegdefessel L., Spiegelman B.M, & Ruas J.L. (2024). Mustn1 is a smooth muscle cell-secreted microprotein that modulates skeletal muscle extracellular matrix composition. Molecular Metabolism, 82, 101912.

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Western Blot Protein Analysis Protocol

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Cells were washed with PBS and lysed in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate; pH 7.4) with Protease Inhibitor Cocktail Set III (EMD Millipore). Lysates were sonicated in a water bath sonicator (Diagenode) at 4 °C for 5 min with 30-s on/off pulses at the low setting. Protein extracts were denatured at 75 °C for 20 min and run at 150 V for 1.5 h on 4-12% NuPAGE Bis-Tris gels in NuPAGE MOPS running buffer (Thermo Fisher). Proteins were transferred to polyvinylidene difluoride membrane using NuPAGE transfer buffer (Thermo Fisher) with 10% methanol. Membranes were blocked in blocking buffer (TBS containing 5% (wt/vol) dry milk powder) for 30 min and probed with primary antibodies in blocking buffer for 16 h at 4 °C. Primary antibodies are described in Supplementary Table 6. Membranes were washed three times with TBS and probed with secondary HRP-conjugated antibodies in blocking buffer for 1 h at room temperature. Signal was detected by Pierce ECL substrate (Thermo Fisher) and exposure to film.

Luo E.C., Nathanson J.L., Tan F.E., Schwartz J.L., Schmok J.C., Shankar A., Markmiller S., Yee B.A., Sathe S., Pratt G.A., Scaletta D.B., Ha Y., Hill D.E., Aigner S, & Yeo G.W. (2020). Large-scale tethered function assays identify factors that regulate mRNA stability and translation. Nature structural & molecular biology, 27(10), 989-1000.

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Proteomic Analysis of Astronaut Muscle

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Muscle lysates of astronauts in RIPA buffer were precipitated in acetone and resuspended in LYSE buffer (PreOmics), heated at 95°C for 5 min and sonicated in a water-bath sonicator (Diagenode) for 15 min. with a 50% duty cycle. Protein concentration in the lysate was adjusted to 2 µg/µl. Proteolytic digestion was carried out by adding 1 µg of endoproteinase LysC and and trypsin per 50 ug of lysate. After overnight digestion at 37°C under continuous shaking, the lysate was acidified to a final concentration of 0.1% TFA and loaded onto StageTip plugs of SDB-RPS. Purified peptides were eluted with 80% acetonitrile-1% ammonia and dried. The dataset consists of technical quintuplicates for the biopsies of two astronauts at three time points.

Murgia M., Ciciliot S., Nagaraj N., Reggiani C., Schiaffino S., Franchi M.V., Pišot R., Šimunič B., Toniolo L., Blaauw B., Sandri M., Biolo G., Flück M., Narici M.V, & Mann M. (2022). Signatures of muscle disuse in spaceflight and bed rest revealed by single muscle fiber proteomics. PNAS Nexus, 1(3), pgac086.

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Ammonium Shock and Cellular Response

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The culture was grown in a modified Sautons’ medium supplemented with 0.5 mM (NH₄)₂SO₄ until reaching an OD of 0.8 at 600 nm. The culture was then split into four fractions. In three of the cultures, (NH₄)₂SO₄ was added to a final concentration of 15 mM, inducing an ammonium shock. After 15 min, the cultures were harvested by centrifugation at 3,000 x g for 10 min at 4°C. The cells were washed twice with PBS and resuspended in 100 μl of PBS containing 1 mM PMSF and either 0.6 mM ADP, or 4.5 mM ATP, and 1.5 mM 2-OG. Then, cultures were lysed using a water bath sonicator (Diagenode), and the lysates were clarified by centrifugation at 11,180 × g for 30 min at 4°C. The resulting cell-free extracts were further centrifuged at 16,000 x g for 1 h at 4°C, and the supernatant, representing the cytosolic fraction, was collected. The pellet, containing the membrane fraction, was washed once with PBS, containing the corresponding additives, and centrifuged again for 30 min at 16,000 g. The membranes were then resuspended in 50 μl of PBS. The samples were analyzed by SDS-PAGE followed by western blotting, performed to detect PII and RpoB, serving as a control.

Ensinck D., Gerhardt E.C., Rollan L., Huergo L.F., Gramajo H, & Diacovich L. (2024). The PII protein interacts with the Amt ammonium transport and modulates nitrate/nitrite assimilation in mycobacteria. Frontiers in Microbiology, 15, 1366111.

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ChIP-seq Analysis of Epigenetic Markers

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The ChIP experiments were performed as previously described.³³ (link)^,³⁴ (link) Briefly, A549 cells were washed with PBS twice and crosslinked with 1% formaldehyde for 10 min. Then, cells were rinsed with ice-cold PBS twice and collected. Cells were collected and resuspended in lysis buffer (50 mM Tris-HCl [pH 8.1], 10 mM EDTA, 1% SDS, 1× protease inhibitor cocktail), and sonicated for 10 cycles at the maximum amplitude using a water bath sonicator (Diagenode) before centrifugation for 10 min. Then, immunoprecipitation was performed using antibodies against CBX2, EZH2, H2AK119ub, and H3K27me3 or non-specific IgG as the negative control. The eluted DNA fragments were purified using a DNA purification kit (QIAquick Spin Kit; Qiagen, Valencia, CA, USA) and subjected to PCR or sent to BGI-Shenzhen (Shenzhen, China) for deep sequencing.³³ (link)

Hu F.F., Chen H., Duan Y., Lan B., Liu C.J., Hu H., Dong X., Zhang Q., Cheng Y.M., Liu M., Guo A.Y, & Xuan C. (2021). CBX2 and EZH2 cooperatively promote the growth and metastasis of lung adenocarcinoma. Molecular Therapy. Nucleic Acids, 27, 670-684.

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Western Blot Analysis of Antioxidant Proteins

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Cells were lysed in ice-cold RIPA buffer with protease inhibitors (Thermo Fisher Scientific, PIA32955) followed by sonication in a water bath sonicator (Diagenode). Protein was quantified using the DC protein assay (Bio-Rad). Lysates were prepared with 6X SDS sample buffer containing 12% (v/v) β-ME (VWR) and separated on Bolt or NuPAGE 4%–12% Bis-Tris gels (Invitrogen). SDS-PAGE separation was followed by transfer to 0.45-μm nitrocellulose membranes (GE Healthcare). The membranes were blocked in 5% non-fat milk in TBS with 0.1% Tween 20 (TBST). For immunoblotting, the following antibodies diluted in 5% milk in TBST were used: KEAP1 (Millipore Sigma, RRID:AB_2921362, 1:2,000), NRF2 (Cell Signaling Technology, D1Z9C, RRID:AB_2715528, 1:1,000), NQO1 (Sigma-Aldrich, RRID:AB_1079501, 1:1,000), GCLC (Sana Cruz Biotechnology, H-5, RRID:AB_2736837, 1:1,000), xCT (Abcam, RRID:AB_778944, 1:1,000), GSR (Santa Cruz Biotechnology, RRID:AB_2295121, 1:1,000), β-actin (Invitrogen AM4302, RRID:AB_2536382, 1:100,000). HRP secondary antibodies used include goat anti-rabbit IgG (Jackson ImmunoResearch Labs, RRID: AB_2313567), goat anti-mouse IgG (Jackson ImmunoResearch Labs, RRID:AB_10015289), and goat anti-rat IgG (Jackson ImmunoResearch Labs, RRID:AB_2338128). Membranes were developed with Clarity ECL substrate (Bio-Rad) or a luminol-based homemade ECL substrate.

DeBlasi J.M., Falzone A., Caldwell S., Prieto-Farigua N., Prigge J.R., Schmidt E.E., Chio I.I., Karreth F.A, & DeNicola G.M. (2023). Distinct Nrf2 Signaling Thresholds Mediate Lung Tumor Initiation and Progression. Cancer Research, 83(12), 1953-1967.

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Western Blot Analysis of Protein Extracts

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Cells were washed with PBS and lysed in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate; pH 7.4) with Protease Inhibitor Cocktail Set III (EMD Millipore). Lysates were sonicated in a water bath sonicator (Diagenode) at 4 °C for 5 min with 30-s on/off pulses at the low setting. Protein extracts were denatured at 75 °C for 20 min and run at 150 V for 1.5 h on 4–12% NuPAGE Bis-Tris gels in NuPAGE MOPS running buffer (Thermo Fisher). Proteins were transferred to polyvinylidene difluoride membrane using NuPAGE transfer buffer (Thermo Fisher) with 10% methanol. Membranes were blocked in blocking buffer (TBS containing 5% (wt/vol) dry milk powder) for 30 min and probed with primary antibodies in blocking buffer for 16 h at 4 °C. Membranes were washed three times with TBST and probed with secondary HRP-conjugated antibodies in blocking buffer for 1 h at room temperature. Signal was detected by Pierce ECL substrate (Thermo Fisher) and imaged on an Azure Biosystems C600 imager.

Xiang J.S., Mueller J.R., Luo E.C., Yee B.A., Schafer D., Schmok J.C., Tan F.E., Rothamel K., McVicar R.N., Kwong E.M., Croker B.A., Jones K.L., Her H.L., Chen C.Y., Vu A.Q., Jin W., Park S.S., Le P., Brannan K.W., Kofman E.R., Li Y., Tankka A.T., Dong K.D., Song Y., Clark A.E., Carlin A.F., Van Nostrand E.L., Leibel S.L, & Yeo G.W. (2022). Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions. Research Square.

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ChIP-qPCR analysis of ABCB1 promoter

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D283 and HD-MB03 cells were cross-linked in 1% paraformaldehyde for 10 min. Cell lysis, shearing, immunoprecipitation and DNA purification were undertaken using a Magna ChIP A/G immunoprecipitation kit (Sigma Aldrich (St. Louis, MO, USA); 17-10085) according to the manufacturer’s instructions. Samples were sonicated using a water bath sonicator (Diagenode, Denville, NJ, USA) to obtain 200–500 bp chromatin fragments. The following antibodies were utilised to pull down protein-DNA complexes, with Immunoglobin (IgG) 1 used as a negative control and histone H3K4Me3 used to confirm active promoter regions: YB-1 (Santa Cruz (Santa Cruz, CA, USA); SC101198); IgG1 (Cell Signalling; 5415); H3K4Me3 (Active Motif (Carlsbad, CA, USA); 61379). To quantify target protein interaction with the gene of interest, qPCR using primers specific to an inverted CCAAT box within the ABCB1 promoter region was undertaken. Primer sequences were as follows: ABCB1 forward (5′ CAT GCT GAA GAA AGA CCA CTG C 3′) and ABCB1 reverse (5′ AGG CTT CCT GTG GCA AAG AG 3′).

Taylor L., Wade P.K., Johnson J.E., Aldighieri M., Morlando S., Di Leva G., Kerr I.D, & Coyle B. (2023). Drug Resistance in Medulloblastoma Is Driven by YB-1, ABCB1 and a Seven-Gene Drug Signature. Cancers, 15(4), 1086.

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Proteomic Profiling of Single Muscle Fibers

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Fibers were microdissected manually under a stereomicroscope from fresh biopsies kept in an ice-cold solution containing K-propionate (150 mM), KH2PO4 (5 mM), Mg Acetate (5 mM), EGTA (5 mM), and DTT (1 mM). They were individually frozen in dry ice in standard Eppendorf tubes. The fiber isolation was completed within 20 min of the muscle biopsy procedure. Fibers were lysed in 20 µl of LYSE (PreOmics), heated at 95°C for 5 min and sonicated in a water-bath sonicator (Diagenode) for 15 min. with a 50% duty cycle. Proteolytic digestion was carried out by adding to the fiber lysate 500 ng of endoproteinase LysC and 500 ng of trypsin in 10 µl of LYSE buffer. After overnight digestion at 37°C under continuous shaking, the lysate was acidified to a final concentration of 0.1% trifluoro-acetic acid (TFA) and loaded onto StageTip plugs of SDB-RPS. Purified peptides were eluted with 80% acetonitrile-1% ammonia and dried. The dataset consists of 233 single fibers, obtained from 10 subjects at three time points.

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Western Blot Analysis of Protein Extracts

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Cells were washed with PBS and lysed in lysis buffer (50 mM Tris-HCl, 100 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate; pH 7.4) with Protease Inhibitor Cocktail Set III (EMD Millipore). Lysates were sonicated in a water bath sonicator (Diagenode) at 4 °C for 5 min with 30-s on/off pulses at the low setting. Protein extracts were denatured at 75 °C for 20 min and run at 150 V for 1.5 h on 4–12% NuPAGE Bis-Tris gels in NuPAGE MOPS running buffer (Thermo Fisher). Proteins were transferred to polyvinylidene difluoride membrane using NuPAGE transfer buffer (Thermo Fisher) with 10% methanol. Membranes were blocked in blocking buffer (TBS containing 5% (wt/vol) dry milk powder) for 30 min and probed with primary antibodies in blocking buffer for 16 h at 4 °C. Membranes were washed three times with TBST and probed with secondary HRP-conjugated antibodies in blocking buffer for 1 h at room temperature. Signal was detected by Pierce ECL substrate (Thermo Fisher) and imaged on an Azure Biosystems C600 imager.

Xiang J.S., Mueller J.R., Luo E.C., Yee B.A., Schafer D., Schmok J.C., Tan F.E., Rothamel K., McVicar R.N., Kwong E.M., Jones K.L., Her H.L., Chen C.Y., Vu A.Q., Jin W., Park S.S., Le P., Brannan K.W., Kofman E.R., Li Y., Tankka A.T., Dong K.D., Song Y., Carlin A.F., Van Nostrand E.L., Leibel S.L, & Yeo G.W. (2022). Discovery and functional interrogation of SARS-CoV-2 protein-RNA interactions. bioRxiv.

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