The 293T cells transfected with Gas-miR01/Gas-miR02/NC mimics were collected, and then the total proteins were extracted using the Cell Lysis Reagent (Roche). Total proteins were separated by 12% SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, USA) by electroblotting. After blocking with 5% skim milk in tris-buffered saline with Tween (TBST) (pH 7.4) for 2 h at room temperature, the protein was detected with an anti-A20 antibody (Cell Signaling Technology, USA) overnight at 4°C. The membrane was washed with TBST, and HRP-labeled (horseradish peroxidase-labeled) mouse anti-rabbit antibody was used as the secondary antibody (Cell Signaling Technology, USA). The signals were detected using the ECL Detection Kit (Advansta).
Cell lysis reagent
Manufactured by Roche
The cell lysis reagent is a solution designed to disrupt and break down the cellular membrane and release the contents of the cell, including proteins, nucleic acids, and other cellular components. It is a key tool used in various laboratory procedures, such as protein extraction and purification, DNA/RNA isolation, and cell fractionation.
Automatically generated - may contain errors
Lab products found in correlation
Atp bioluminescence assay kit hs 2, by Roche (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Anti a20 antibody, by Cell Signaling Technology (1 mentions)
Ecl detection kit, by Advansta (1 mentions)
Fluostar optima microplate reader, by BMG Labtech (1 mentions)
Luciferase reagent, by Roche (1 mentions)
Protein assay reagent, by Bio-Rad (1 mentions)
Biotek synergy 2, by Agilent Technologies (1 mentions)
3 protocols using cell lysis reagent
1
Western Blot Analysis of A20 Protein
2
Measurement of Cellular and Mitochondrial ATP
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Cellular and mitochondrial ATP contents were measured in freshly prepared whole cell lysates and mitochondrial suspensions from left ventricles (LVs) according to manufacturer's instructions using the ATP Bioluminescence Assay Kit HS II (Roche).26 , 27 Freshly excised LVs were chopped and homogenized using a hand‐driven glass/Teflon potter Elvehjem homogenizer (Wheaton) in cell lysis reagent provided in the kit on ice. Fresh mitochondria suspensions were collected as described above and homogenized in cell lysis reagent (Roche) using this method on ice. Prepared whole cell and mitochondria lysates were then left at RT for 5 minutes following boiling at 100°C for 2 minutes to extract ATP according to manufacturer's instructions (Roche). The lysates were then centrifuged at 10 000g for 1 minute and supernatants were collected to measure the ATP level. ATP content was measured immediately after addition of luciferase reagent (Roche) by automated injection and luciferase‐driven bioluminescence was measured on a FLUOstar OPTIMA microplate reader (BMG Labtech). ATP levels in samples were then normalized to protein content as measured by Bio‐Rad protein assay reagent.26
3
Quantification of Cellular ATP Levels
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ATP levels were quantified using an ATP Bioluminescence assay kit HS II (Roche Diagnostics) according to the manufacturers’ instructions. Cells were lysed with cell lysis reagent (Roche Diagnostics) and 50 μl lysates were mixed with 150 μl luciferase assay reagent (Roche Diagnostics). The luminescence was detected using a plate reader (Biotek Synergy 2; BioTek Instrument, Inc., Winooski, VT, USA). The protein concentrations of the samples were determined by a BCA assay. The ATP concentrations of the samples were calculated using an ATP standard and normalized against the amount of protein in the samples.
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