Liver samples were homogenized in lysis buffer [10 mmol/L Tris, pH 7.5, 150 mmol/L NaCl, 1% Triton X-100, 1 mmol/L phenylmethylsulfonyl fluoride, 0.2 mmol/L sodium orthovanadate, 0.5% Nonidet P-40 containing protease and phosphatase inhibitor cocktail tablets (Roche) at 4°C for 30 minutes. Protein was quantified using the protein assay (#21011, Intron, Korea), and proteins were resolved on 7.5%-10% SDS/ PAGE gels. The membranes were blocked for 1 hour in tris-buffered saline (TBS) containing 0.1% Tween-20 (TBS-T) and 5% skim milk and then incubated overnight at 4°C with a primary antibody (see above) in the same buffer. The blots were then washed three times in TBS-T for 15 minutes to remove excess antibody, and then the membranes were incubated for 2 hours with secondary antibodies in TBS-T+ 5% (g/vol) skim milk: (AE-1475 Bioss, Wobrun, MA, USA) or anti-mouse (bs-0296G). Following three washes in TBS-T, proteins were detected with ECL solution (XLS025-0000, Cyanagen, bologna, Italia) and Chemi Doc (Fusion Solo, Vilber Lourmat, France).
Xls025 0000
Manufactured by Cyanagen
The XLS025-0000 is a laboratory equipment device. It is designed for performing specific laboratory functions. The core function of the XLS025-0000 is to facilitate certain scientific procedures and processes within a laboratory setting.
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3 protocols using xls025 0000
1
Western Blot Analysis of Liver Proteins
2
Western Blot Analysis of Total Liver and Hep3B Cells
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Total liver and hep3B cell samples were homogenized by protein lysis buffer [T-PER (#78510, Thermo)] containing proteinase inhibitor (PMSF). Homogenized samples were quantified by Bradford assay with PRO-Measure solution (Intron, #21011) and proceeded to protein electrophoresis (SDS-PAGE) after 5 min of boiling in 100 °C. Gels were blotted by wet transfer with Bio-Rad Power Pac in 350 mA. Membranes were blocked for 1 hour in Skim milk and incubated with a primary antibody (see above) for overnight, 4 °C. After 3 times of washing with PBS-T or TBS-T, membranes were incubated with secondary antibodies (AE-1475 goat anti-rabbit, BS-0296G-HRP goat anti-mouse, Bioss) for 4 hours diluted with 1:5000 in skim milk or BSA. Results were detected with ECL solution (XLS025-0000, Cyanagen) and Chemi Doc (Fusion Solo, Vilber Lourmat).
3
Western Blot Analysis of Protein Samples
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Both protein samples of cerebrums and DBT cell was extracted by using protein lysis buffer, called T-PER reagent (78510, Thermo Fisher scienti c) and quanti ed by Bradford assay with PRO-Measure solution (#21011, Intron). The samples were run SDS-PAGE electrophoresis on 10, 12% polyacrylamide gels and transferred to membrane. And the membranes were blocked with 30mg/ml BSA100 (9048-46-8, LPS solution), diluted TBS-T buffer (04870517TBST4021, LPS solution). Primary antibodies were operated during overnight in 4℃. Following the step, the membranes were washed with TBS-T and secondary antibodies were operated in identical way. Results were detected with ECL solution (XLS025-0000, Cyanagen) and Chemi Doc (Fu-sion Solo, VilberLourmat). The primary and secondary antibody information are as for Table.
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