The HEK 293 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin until 90% confluence as previously described.7 (link) After washing with PBS three times, cell pellets were resuspended in lysing buffer (100 mM sodium chloride, 50 mM sodium phosphate, 10% glycerol, 2 mM ATP, 1 mM TCEP, 10 mM MgCl2, 1x protease inhibiter (Roche), 1× phosphatase inhibitor, and 0.5% NP-40, pH 7.5). The cell debris was removed by centrifugation at 13,000 rpm for 15 min at 4 °C. The supernatant was collected and adjusted to 1 mg/ml, which was subjected to cross-linking with 2 mM DSSO at room temperature for 1 h. The cross-linking reaction was quenched with 50 mM ammonium bicarbonate for 10 min.
Protease inhibiter
Manufactured by Roche
The Protease Inhibiter is a laboratory equipment designed to inhibit the activity of proteases, which are enzymes that break down proteins. Its core function is to preserve the integrity of protein samples during various analytical and research procedures.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor, by Roche (1 mentions)
Chemidoc analyzer, by Bio-Rad (1 mentions)
Bioruptor, by Cosmo Bio (1 mentions)
Anti actin antibody, by Thermo Fisher Scientific (1 mentions)
Anti h3 antibody, by Abcam (1 mentions)
Ecl prime western blotting detection reagent, by GE Healthcare (1 mentions)
Trans blot turbo, by Bio-Rad (1 mentions)
2 protocols using protease inhibiter
1
Crosslinking of HEK 293 Cell Lysates
2
Western Blot Analysis of Cellular Proteins
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Whole-cell extracts were prepared in cell lysis buffer (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.5 mM EDTA, 0.5% NP-40, protease inhibiter [Roche]) with sonication (30 s ON/90 s OFF × 6 times) using Bioruptor (Cosmobio). Protein samples were separated by SDS-PAGE and transferred onto a PVDF membrane using a semi-dry transfer system (Trans-Blot Turbo, BioRad). The transferred membrane was blocked with TBS-T (10 mM Tris-HCl [pH 8.0], 150 mM NaCl, 0.05% Tween20) containing 5% BSA. The membrane was incubated 1 hour in a primary antibody diluted with Can Get Signal solution 1 (TOYOBO), and washed with TBS-T three times. The membrane was then incubated for one hour in a secondary antibody diluted with Can Get Signal solution 2 (TOYOBO). Washing three times with TBS-T, signals were developed with ECL prime western blotting detection reagents (GE Healthcare), and subsequently detected using the Chemidoc analyzer (BioRad). Primary antibodies were used at the following dilution rate. Anti-actin antibody (Thermo Fisher Scientific, MS-1295-P1): 1/1000. Anti-H3 antibody (Abcam, ab1791): 1/10,000. Anti-GFP antibody (MBL, 598): 1/5000. Anti-rabbit IgG (Promega, W4011) or anti-mouse IgG (Promega, W4021) were used as secondary antibodies at 1/10,000 dilution.
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