For each genetic background, MTs from >70 flies were dissected under Schneider's medium and subsequently transferred to 100 μl RIPA buffer (150 mM NaCl, 10 mM TrisHCL ph 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% SDS) with HALT protease inhibitor (Thermo Fisher Scientific). Samples were homogenized using a Microson XL2000 sonicator (Misonix Inc. NY, USA), and ultra centrifuged (17,000g) at 4 °C for 5 min, before transferring the supernatant to fresh tubes. Protein contents were measured using the Bradford assay (Precision Red, Cytoskeleton). Approximately 15 μg protein from each sample was electrophoresed on a NuPage 4–12% Bis-Tris gel (Invitrogen), and blotted onto nitrocellulose using the Novex system (Invitrogen). Blots were probed with monoclonal anti-Fas2 34B3 (1:500) and anti-GAPDH (1:1,000, Abcam, UK, cat# ab125247) antibodies, and developed with DyLight800-conjugated goat anti-mouse IgG (1:10,000, Thermo Fisher Scientific, cat# SA5-35521) and Alexa Fluor 680-conjugated goat anti-rabbit IgG (1:10,000, Thermo Fisher Scientific, cat# A-21109) secondary antibodies, using a Li-Cor CLx Odyssey. Fas2 expression was quantified relative to GAPDH levels using Image Studio Lite software (Li-Cor Biosciences), and the data represented as mean±s.e.m. based on three independent experimental repeats. The full scan is provided in Supplementary Fig. 5 .
Microson xl 2000 sonicator
Manufactured by Bioventus
Sourced in United States
The Microson XL-2000 is a sonicator device that utilizes ultrasonic waves to disrupt samples. It is designed for various laboratory applications that require sample homogenization, cell lysis, or emulsification.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktails 2 and 3, by Merck Group (2 mentions)
Protein assay, by Bio-Rad (2 mentions)
P1 probe, by Bioventus (2 mentions)
Novex system, by Thermo Fisher Scientific (1 mentions)
Image studio lite software, by LI COR (1 mentions)
Nupage 4 12 bis tris gel, by Thermo Fisher Scientific (1 mentions)
Halt protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Anti gapdh, by Abcam (1 mentions)
Phosphate inhibitor cocktail 3, by Merck Group (1 mentions)
Lipofectamine reagent, by Thermo Fisher Scientific (1 mentions)
Plus reagent, by Thermo Fisher Scientific (1 mentions)
Phosphate inhibitor cocktail 2, by Merck Group (1 mentions)
Halt protease inhibitor cocktail, by Merck Group (1 mentions)
Phosphatase inhibitors 2 and 3, by Merck Group (1 mentions)
Halt protease inhibitor, by Merck Group (1 mentions)
Kh2po4, by Merck Group (1 mentions)
Na2hpo4, by Merck Group (1 mentions)
Potassium chloride (kcl), by Merck Group (1 mentions)
9 protocols using microson xl 2000 sonicator
1
Quantitative Western Blot Analysis of Fas2
2
Cell Lysis and Protein Extraction
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Cells were washed once with 1X phosphate buffered saline (PBS) and lysed in 300 μl TLB-sodium dodecyl sulphate (SDS) buffer [50 mM Tris, 150 mM NaCl, 10 mM ethylenediaminetetraacetic acid (EDTA), 0.5% SDS, 0.5% Triton-X, pH 7.2] supplemented with 10 μl/ml each of the Halt protease inhibitor cocktail (Pierce) and Phosphatase inhibitor cocktails 2 and 3 (Sigma). Harvested cells were sonicated three times for 10 s each at 12 W using a Microson XL-2000 sonicator (Misonix). Cell lysates were collected after centrifugation at 14 000 rpm for 15 min at 4°C.
3
Cell Lysis and Protein Quantification
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Cells were washed once with 1X phosphate buffered saline (PBS) and lysed in 300 μl TLB-sodium dodecyl sulphate (SDS) buffer [50 mM Tris, 150 mM NaCl, 10 mM ethylenediamine-tetraacetic acid (EDTA), 0.5 % SDS, 0.5 % Triton-X, pH 7.2] supplemented with 10 μl/ml each of the Halt protease inhibitor cocktail (Pierce) and Phosphatase inhibitor cocktails 2 and 3 (Sigma). Harvested cells were sonicated three times for 10 s each at 12 W using a Microson XL-2000 sonicator (Misonix). Cell lysates were collected after centrifugation at 14,000 rpm for 15 min at 4 °C. Total protein concentration was calculated using the Bio-Rad Protein Assay.
4
Chromatin Immunoprecipitation (ChIP) of ERα
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ChIP analysis was performed using the EZ-ChIP system (Merck Millipore, 17–371) according to the manufacturer’s instructions. Briefly, MCF-7 cells were treated with E2 for 24 h, and then fixed in 37% formaldehyde, resuspended in lysis buffer, and sonicated 10 × 10 s (amplitude 5%) in a Microson XL-2000 sonicator (Misonix). Supernatants were pre-cleared with Protein G agarose beads and 1% of the input was reserved for later analysis. Lysates (from 1 × 106 cells for each sample) were then immunoprecipitated overnight at 4 °C using antibodies to ERα or normal mouse IgG. DNA–protein complexes were captured by incubation for 1 h at 4 °C with Protein G agarose beads. Complexes were washed, resuspended in elution buffer and incubated for 5 h at 65 °C to reverse cross-links. DNA was then isolated, and EREs were detected with PCR. Primers were as follows: ERE (−726 to −704 bp from the transcriptional start site) in the 5′ upstream region of PPP1CA; 5′-TCAAAAGCTAATTATGGGGCC-3′ and 5′-TCAAGCGATTCTCCTGCCTCA-3′.
5
Nucleoli Isolation from Cell Lines
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Nucleoli were isolated from HT1080, uninduced T19, and 30- or 32-hour induced T19 cells using a previously described method [28] subsequently modified by the Lamond lab [29] (link). Briefly, ∼108 cells were harvested, washed with cold PBS, resuspended in 5–7 mL of buffer (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM dithiothreitol), and dounce homogenized on ice (tight pestle) until nuclei were released. Nuclei were gently pelleted, resuspended in 3 mL of Sucrose 1 (0.25 M sucrose, 10 mM MgCl2), and layered over Sucrose 2 (0.35 M sucrose, 0.5 mM MgCl2) before centrifugation at 1430×g for 5 minutes. The pellet was resuspended in 3 mL Sucrose 2 before sonication with a Misonix Microson XL 2000 sonicator at power 10 with 5 second bursts on ice until nuclei were broken open and nucleoli were distinct. The sonicated solution was layered over 3 mL Sucrose 3 (0.88 M sucrose, 0.5 mM MgCl2) and centrifuged at 2800×g for 10 minutes before resuspension in Sucrose 2.
6
Transfection and Protein Extraction Protocol
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Two million cells seeded 16–18 h prior to transfection in T75 flasks. 6 μg of appropriate expression construct or appropriate empty vector (control) were transfected with 24 μl Lipofectamine reagent (Life Technologies) and 12 μl Plus reagent (Life Technologies) (17 (link)). For coexpression experiments (Supplementary Figures S4 and S7), 4 μg of appropriate expression constructs were transfected separately and together, with empty vector used as filler to bring total DNA transfected to 8 μg. Approximately 24 h post-transfection, cells were washed 1× with phosphate buffered saline (PBS) and then harvested in 500 μl Total lysis buffer (TLB: 50 mM Tris, 150 mM NaCl, 10 mM EDTA, 0.5% SDS, 0.5% Triton-X, pH 7.2) supplemented with 10 μl/ml of Halt protease inhibitor cocktail, phosphate inhibitor cocktail 2, and phosphate inhibitor cocktail 3 (Sigma). After one round of freeze (−80°C) /thaw on ice, cells were sonicated with a Microson XL-2000 sonicator (Misonix) 3× (10 s sonication/10 s rest on ice), and cell lysates were then centrifuged at 4°C at 14 000 rpm for 15 min. Protein concentrations of cleared cell lysates were determined using BioRad protein assay (Bradford method).
7
Fish oil-in-water emulsion stabilized by potato peptides
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Peptides (0.2 wt.%) were dissolved in 10 mM sodium acetate -10 mM imidazole buffer (pH 7). The peptide solutions were shaken (100 rpm) overnight at room temperature to allow complete solubilization and rehydration of the peptides (when possible). Primary homogenization was done by adding the fish oil to the peptide-buffer solution and mixing at 18,000 rpm for 30 s by using a POLYTRON® PT1200E (Kinematic Inc., New York, USA).
Secondary homogenization was done using a Microson XL2000 sonicator equipped with a P1 probe (Misonix, Inc., New York, USA). Emulsions were homogenized at an amplitude of 75% (maximum amplitude of 180 µm), running 2 passes of 30 s with a break of 1 min between passes. During sonication the emulsions were surrounded by iced water to minimize the increase in temperature. A total amount of 2 g of 5 wt.% fish oil-in-water emulsion stabilized with 0.2 wt.% potato peptides was produced.
Secondary homogenization was done using a Microson XL2000 sonicator equipped with a P1 probe (Misonix, Inc., New York, USA). Emulsions were homogenized at an amplitude of 75% (maximum amplitude of 180 µm), running 2 passes of 30 s with a break of 1 min between passes. During sonication the emulsions were surrounded by iced water to minimize the increase in temperature. A total amount of 2 g of 5 wt.% fish oil-in-water emulsion stabilized with 0.2 wt.% potato peptides was produced.
8
Fractionation of Nuclear and Cytoplasmic Proteins
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The processing of nuclear and cytoplasmic fractions was performed as previously described (83 (link),84 (link)) Specifically, HeLa cells were washed with 1× PBS (137 mM NaCl (Sigma S9888), 2.7 nM KCl (Sigma P4505), 10 mM Na2HPO4 (Sigma S3264) and 2 mM KH2PO4 (Sigma P9791), pH 7.4) and harvested using 500 μl of TLB (50 mM Tris, 150 mM NaCl, 10 mM EDTA, TritonX-100 0.5% v/v, Halt Protease inhibitor 10 μl/ml, phosphatase inhibitors 2 and 3 (Sigma), pH 7.2) buffer per T75 flask. The samples were centrifuged at 13 000 rpm for 15 min at 4°C. The supernatant was transferred to a new microcentrifuge tube (this is the cytoplasmic fraction). The remaining nuclear pellet in the microcentrifuge was gently washed three times with 200 ul of TLB buffer. The nuclear pellets were resuspended in TLB and the nuclear lysate samples were sonicated with a Microson XL-2000 sonicator (Misonix) 4× (6 s sonication/10s rest on ice). Samples were centrifuged at 21 130g for 15 min at 4°C. The resulting supernatant (the nuclear fraction) was then transferred to a new microcentrifuge tube. The protein concentration for each sample was determined using 595 nm wavelength OD values against a bovine serum albumin (BSA) standard.
9
Fish oil-in-water emulsion stabilized by potato peptides
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Peptides (0.2 wt.%) were dissolved in 10 mM sodium acetate -10 mM imidazole buffer (pH 7). The peptide solutions were shaken (100 rpm) overnight at room temperature to allow complete solubilization and rehydration of the peptides (when possible). Primary homogenization was done by adding the fish oil to the peptide-buffer solution and mixing at 18,000 rpm for 30 s by using a POLYTRON® PT1200E (Kinematic Inc., New York, USA).
Secondary homogenization was done using a Microson XL2000 sonicator equipped with a P1 probe (Misonix, Inc., New York, USA). Emulsions were homogenized at an amplitude of 75% (maximum amplitude of 180 µm), running 2 passes of 30 s with a break of 1 min between passes. During sonication the emulsions were surrounded by iced water to minimize the increase in temperature. A total amount of 2 g of 5 wt.% fish oil-in-water emulsion stabilized with 0.2 wt.% potato peptides was produced.
Secondary homogenization was done using a Microson XL2000 sonicator equipped with a P1 probe (Misonix, Inc., New York, USA). Emulsions were homogenized at an amplitude of 75% (maximum amplitude of 180 µm), running 2 passes of 30 s with a break of 1 min between passes. During sonication the emulsions were surrounded by iced water to minimize the increase in temperature. A total amount of 2 g of 5 wt.% fish oil-in-water emulsion stabilized with 0.2 wt.% potato peptides was produced.
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