ChIPs were performed as described in Whyte et al.31 (link) using ~100 × 106 HeLa-LTR-Luciferase cells as starting material with the exception of an additional nuclei purification step. Briefly, cells were thawed and resuspended in 1 ml Buffer A (0.3 M SUCROSE, 60 mM KCl, 15 mM NaCl, 5 mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2 mM PMSF), 1 ml Buffer B (0.3 M SUCROSE, 0.2% NP40, 60 mM KCl, 15 mM NaCl, 5 mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2 mM PMSF) was then added and incubated for 7 min on ice, laid over a 8 ml cushion of Buffer C (1.2 M SUCROSE, 0.2% NP40, 60 mM KCl, 15 mM NaCl, 5 mM MgCl, 0.1 mM EGTA, Tris-HCl pH=7.5, 0.2 mM PMSF) and spinned for 20 min at 3000, r.p.m. on 4 °C. Pelleted nuclei were resuspended in 3-ml lysis buffer, incubated for 1 h, sonicated (Misonix sonicator (Misonix) with the following settings: micro tip, 30 s on, 2 min off, amplitude 70, 7 min total sonication time) and processed as described in Whyte et al.31 (link) qPCR was carried out in the LightCycler480 (Roche) with a 15 min DNA denaturation step at 95 °C, followed by 50 cycles of 15 s at 95 °C, 30 s at 58 °C and 30 s at 72 °C. PCR measurements were performed in duplicate. Enrichments are expressed in percentage of input ((2−ΔCT)*100/(Vol IP/Vol input)). Averages and standard deviations of experimental replicates are shown in the figures.
Misonix sonicator
Manufactured by Bioventus
The Misonix sonicator is a lab equipment product designed for cell lysis and homogenization. It utilizes high-frequency sound waves to disrupt cellular structures and release the contents of cells. The sonicator is a versatile tool used in various applications, such as protein extraction, DNA shearing, and sample preparation for analysis.
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3 protocols using misonix sonicator
1
ChIP-seq Protocol for HeLa Cells
2
Waste Sludge to Lipid-Rich Algae
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Example 4
In one example, fresh waste activated sludge was first diluted with tap water until the solid concentration was 7.6 g/L. In 300 mL sludge, the pH was adjusted to 10 by NaOH and then the sludge was sonicated for 10 min by a MISONIX® sonicator. After this alkaline-sonication pretreatment, the organics in supernatant increased to 2 g/L. The sonicated sludge was centrifuged at 500 g for 10 min and the supernatant was acidified to a pH of 4 and fed to algae. After treatment with algae for 2 days, the lipid concentration increased from 0.07 g/L to 0.17 g/L and total organic mass reduction in this sludge-algae system was 42%.
3
Protein Extraction and Western Blot Analysis
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Transfected cells were treated with 200 µl protein lysis buffer (0•15 M-NaCl, 5 mM-EDTA (pH 8), 1 % Triton X100, 10 mM-Tris-Cl, 5 mM-DTT and 1 μM-PMSF). Samples were sonicated using a Misonix Sonicator (Misonix). Cells were centrifuged at 18 800 g for 15 min at 4°C. Protein concentration was determined using a modified Lowry assay (28) and was measured using a Multiskan Spectrum spectrophotometer (Thermo) at an absorbance of 740 nm. Cell lysates containing 40 µg of protein were electrophoresed on Criterion's pre-cast TGX Stain-free 8-16 % polyacrylamide gels (BIORAD).
The gel was equilibrated in transfer buffer containing 20 % (v/v) methanol for approximately 10 min. Proteins were transferred to a polyvinylidine fluoride membrane at 4°C for 1 h. The membrane was removed and placed in blocking buffer (5 % skim milk powder in TBST containing 0•1 % Tween 20) for 1 h. The membrane was incubated overnight at 4°C with primary antibody (1:5000 mouse polyclonal anti-MTR (Santa Cruz Biotechnology) or 1:1000 mouse monoclonal anti-MTHFD1 (Abnova)). An anti-mouse IgG secondary antibody conjugated to horseradish peroxidase was used. Membranes were washed and developed in Enhanced Chemiluminescence solution (BIORAD), and the image was acquired using an Alpha Innotech Fluor Chem HD 2 Imaging System.
The gel was equilibrated in transfer buffer containing 20 % (v/v) methanol for approximately 10 min. Proteins were transferred to a polyvinylidine fluoride membrane at 4°C for 1 h. The membrane was removed and placed in blocking buffer (5 % skim milk powder in TBST containing 0•1 % Tween 20) for 1 h. The membrane was incubated overnight at 4°C with primary antibody (1:5000 mouse polyclonal anti-MTR (Santa Cruz Biotechnology) or 1:1000 mouse monoclonal anti-MTHFD1 (Abnova)). An anti-mouse IgG secondary antibody conjugated to horseradish peroxidase was used. Membranes were washed and developed in Enhanced Chemiluminescence solution (BIORAD), and the image was acquired using an Alpha Innotech Fluor Chem HD 2 Imaging System.
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