Fibrils were diluted into 1X detergent buffer [0.5% (v/v) Nonidet P-40, 0.5% (w/v) sodium deoxycholate in PBS] containing PK (Thermo Scientific #EO0491) at a concentration of 50 μg/mL (PK:protein ratio of 1:1) and then incubated at 37 °C for 1 h with continuous shaking. Digestions were halted by adding PMSF to a final concentration of 4 mM and then samples were ultracentrifuged at 100,000x g for 1 h at 4 °C. The supernatant was discarded, and the pellets were resuspended by boiling in 1X Bolt lithium dodecyl sulfate (LDS) sample buffer (Thermo Scientific) containing 2.5% (v/v) β-mercaptoethanol (“loading buffer”). Samples were then analyzed by SDS-PAGE followed by silver staining.
Eo0491
Manufactured by Thermo Fisher Scientific
The EO0491 is a laboratory equipment product from Thermo Fisher Scientific. It is designed for general laboratory use. The core function of this product is to provide a controlled environment for various laboratory applications.
Automatically generated - may contain errors
Lab products found in correlation
Bolt lithium dodecyl sulfate sample buffer, by Thermo Fisher Scientific (2 mentions)
Protease inhibitor, by Roche (1 mentions)
Mak034, by Merck Group (1 mentions)
Af3628, by R&D Systems (1 mentions)
A21432, by Thermo Fisher Scientific (1 mentions)
C1 confocal microscope, by Nikon (1 mentions)
Vectashield mounting medium, by Vector Laboratories (1 mentions)
Am9260g, by Thermo Fisher Scientific (1 mentions)
Am9759, by Thermo Fisher Scientific (1 mentions)
Minielute purification kit, by Qiagen (1 mentions)
7 protocols using eo0491
1
Proteinase K Digestion of Fibrils
2
Purification and Proteinase K Digestion of Spermatogenic Cells
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Spermatogenic cells were purified by the above method and subjected to in situ proteinase K digestion41 (link). In brief, after two rinses with ice-cold PBS, one lot was incubated in 4 µg/ml proteinase K (EO0491, Thermo Scientific) in KHM buffer (110 mM KOAc, 20 mM Hepes, pH 7.4 and 2 mM MgCl2) for 45 min at room temperature. The second lot was permeabilized with 24 µM of ice-cold digitonin in KHM for 15 min followed by 4 µg/ml proteinase K digestion in KHM for 45 min at room temperature. The third lot was incubated with 4 µg/ml proteinase K in KHM containing 0.5% Triton X-100 for 45 min. Subsequently, PMSF was added to all lots to a final concentration of 40 μg/ml. Cells were then washed in KHM and lysed in 0.4% SDS, 2% Triton X-100, 400 mM NaCl, 50 mM Tris–HCl, pH 7.4, 40 μg/ml PMSF, 1 mM dithiothreitol, and protease inhibitors (04693132001, Roche) by passing through a 22-gauge needle before centrifugation for 10 min at 16,000 × g. Proteins were then probed with specific antibodies by western blotting.
3
Proteinase K Digestion of Fibrils
4
Mitochondrial Protein Solubilization and Digestion
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Isolated mitochondria were resuspended in isotonic buffer (250 mM sucrose, 1 mM EDTA, 10 mM HEPES-KOH pH 7.4) and differentially solubilised in a range of digitonin concentrations or 1% Triton X for 10 min on ice. They were then digested in 100 μg/ml PK (Thermo Scientific EO0491) on ice for 30 min before the digestion was stopped in 8 mM PMSF. Samples were analyzed by SDS-PAGE and immunoblotting.
5
Quantification of CoA Release from Purified TMEM120A
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HsTMEM120A was purified using the same protocol as described above. To release any bound CoA substrate from the protein, HsTMEM120A was subjected to protease digestion with 1 mg/ml proteinase K at 37°C for 1 hr (Thermo Scientific; EO0491). 0.2% sodium dodecyl sulfate was added to the digestion solution to stimulate the activity of proteinase K. After digestion, proteinase K was denatured by incubating the sample at 70°C for 7 min.
CoA levels in the protein solution after proteinase K digestion were quantified using a commercial CoA assay kit according to the manufacturer’s protocol (Sigma-Aldrich; MAK034). CoA concentration is determined by an enzymatic assay, in which a colored product is developed and the colorimetric (OD at 570 nm) or fluorometric (Ex = 535 nm/Em = 587 nm) measurement of the product is proportional to the amount of CoA in the sample. We used fluorometric measurement in our assay for CoA quantification and its concentration was determined by comparing to a standard curve plotted using the pure CoA standard in the assay.
CoA levels in the protein solution after proteinase K digestion were quantified using a commercial CoA assay kit according to the manufacturer’s protocol (Sigma-Aldrich; MAK034). CoA concentration is determined by an enzymatic assay, in which a colored product is developed and the colorimetric (OD at 570 nm) or fluorometric (Ex = 535 nm/Em = 587 nm) measurement of the product is proportional to the amount of CoA in the sample. We used fluorometric measurement in our assay for CoA quantification and its concentration was determined by comparing to a standard curve plotted using the pure CoA standard in the assay.
6
Immunofluorescent Imaging of Vascularization
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Paraformaldehyde-fixed tumours, sWAT and iBAT tissue samples were digested with 20 μg ml−1 proteinase K (EO0491, Thermo Fisher Scientific) in a 10 mM Tris-HCl buffer (pH 7.4) for 5 min and blocked with 3% skim milk, followed by staining overnight at 4 °C with goat anti-mouse CD31 antibodies (1:200, AF3628, R&D systems). After rigorous rinsing with PBS, blood vessels were detected with donkey anti-goat Alexa Fluor 555-labelled secondary antibodies (1:300, A21432, Thermo Fisher Scientific), mounted in Vectashield mounting medium (Vector Laboratories), and stored at −20 °C in the dark before microscopy examination using the Nikon C1 confocal microscope (Nikon). The images were recorded using the camera of a Nikon C1 confocal microscope using the EZ-C1 v.3.91 software (Nikon). Captured images were further analysed using Adobe Photoshop CS5 extended software.
7
Genomic DNA Extraction from FFPE and Frozen Nuclei
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For FFPE-ATAC samples, single nuclei were isolated following the nuclei isolation protocol stated in the section on nuclei isolation from FFPE tissue sections. For frozen samples, nuclei were isolated following the nuclei isolation protocol in the section on standard ATAC-seq on frozen tissue. For genomic DNA purification, 1 million isolated nuclei were spined down at 3000g for 10 min and then resuspended with 100 µL of lysis buffer (50 mM Tris-HCl at pH 7.5 [Invitrogen 15567027], 1 mM EDTA [Invitrogen AM9260G], 1% SDS [Invitrogen 1553-035], 200 mM NaCl [Invitrogen AM9759], and 200 µg/mL Proteinase K [Thermo Fisher Scientific EO0491]). Nuclei suspension was incubated overnight at 65°C with 1200 rpm shaking in a heat block. On the next day, the mixture was purified with a Qiagen MiniElute purification kit (Qiagen 28004) and eluted in 20 µL of elution buffer. Purified genomic DNA was measured and was run on a 1.5% agarose gel (Lonza 50004) to check size distribution.
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