Horseradish peroxide conjugated secondary goat anti rabbit or anti mouse antibodies

The proteins from whole extracts (typically 20–40 μg) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose membrans (Schleicher & Schuell, Dassel, Germany) by a Mini Trans-Blot apparatus BioRad (Hercules, CA, USA). The membranes were washed in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), and blocked with TBST supplemented with 5% nonfat dry milk. After the membranes were incubated overnight with different primary antibodies in TBST and 5% nonfat dry milk. The day after, the membranes were incubated with horseradish peroxide-conjugated secondary goat anti-rabbit or anti-mouse antibodies, conjugated with horseradish peroxidase (BioRad). Enhanced chemiluminescence detection reagents were used as a detection system (ECL) according to the manufacturer’s instructions (Amersham Biosciences, Little Sharfont, UK).

Typically, we employed 20–40 μg of total extracts for immunoblotting [56 (link)]. Proteins from cell preparations were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred onto nitrocellulose sheets (Schleicher & Schuell, Dassel, Germany) by a Mini Trans-Blot apparatus (BioRad, Hercules, CA, USA). Membranes were washed in TBST (10 mM Tris, pH 8.0, 150 mM NaCl, 0.05% Tween 20), and were blocked with TBST supplemented with 5% nonfat dry milk. Then the membranes were incubated with different primary antibodies in TBST and 5% nonfat dry milk, washed, and incubated with horseradish peroxide-conjugated secondary goat anti-rabbit or anti-mouse antibodies, conjugated with horseradish peroxidase (BioRad). Enhanced chemiluminescence detection reagents were used as a detection system (ECL), according to the manufacturer’s instructions (Amersham Biosciences, Little Chalfont, UK).