Cells were lysed using lysis buffer (50 mM HEPES (pH 7.5), 100 mM NaCl, 50 mM KCl, 1% Triton X-100, 5% glycerol, 0.5% NP-40, 2 mM MgCl2, 1 μM DTT, cocktail and PMSF) followed by sonication and centrifugation at 4 °C. Extracts were quantified using the BCA metho...
Lastly, the digested peptides were collected in a new tube via centrifugation. Sequential elution was performed using 50 mM HEPES and 0.5 M NaCl. Peptide concentrations were measured by tryptophan fluorescence assay23. The peptides obtained from the hTEC samples were subjected to TMT labeling...
Cell pellets were resuspended in LDOX lysis buffer (20 mM potassium phosphate, 200 mM NaCl, pH 7.4, 0.01 mg ml−1 DNAseI) to an OD600 of 140 and lysed by two rounds of sonication for 2 min at 50% amplitude with 2 s intervals using a Sonopuls (Bandelin) sonicator. ...
For purification, the bacterial pellet was resuspended in the lysis buffer (50 mM Na2HPO4/NaH2PO4 (pH 8.0), 500 mM NaCl, 0.01 mM ZnCl2, 10% glycerol). Protein lysis was performed via SLM AMINCO French Press (Thermo Fisher Scientific), and the protein solution was centrifuged (40,000g, 45...
We used western blot analysis to detect protein expression levels of FOSL2 and tight junction-related proteins in in vitro BTB models. Cells were harvested and lysed in Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime Institute of Biotechnology, Jiangsu, China) mixed with PMSF (protease...
Cell pellets were suspended in lysis buffer (50 mM Tris–HCl (pH 8.0), 100 mM NaCl, 5 mM DTT, 1 mM PMSF) and lysed by FastPrep FP120 instrument (BIO101, ThermoSavent) by 5 rounds of 30 second at speed 6.5 followed by 2 min on ice. Cell debris was removed by centrifugation at ...
Western blot analysis. Cells were lysed in either NP40 lysis buffer [1% NP40, 50 mmol/L Tris (pH 7.4), 150 mmol/L NaCl] with Complete Protease Inhibitor Cocktail tablets (Roche Diagnostics) or Triton X-100 lysis buffer [1% Triton X-100, 50 mmol/L TBS (pH 7.4), 10 mmol/L EDTA ...
Acetate metabolism in Escherichia coli plays an important role in the control of the central metabolism and in bioprocess performance. The main problems related to the use of E. coli as cellular factory are i) the deficient utilization of carbon source d
Abnormal penile foreskin development in hypospadias is the most frequent genital malformation in male children, which has increased dramatically in recent decades. A number of environmental factors have been shown to be associated with hypospadias develo
Macrophages were harvested in lysis buffer (50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 10% glycerol, 0.5% Triton X-100, 1 mM PMSF, and complete protease inhibitor cocktail) and incubated on ice for 30 min, followed by centrifugation at 6000 gfor 15 min at 4 °C. The su...