Amersham ecltm blocking agent

Fetal hepatoblasts infected with mock or Mist1-overexpressing retrovirus were washed with PBS and lysed with RIPA buffer (20 mM TrisHCl pH7.4, 150 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% sodium deoxycholate, 0.1% sodium dodecyl sulfate [SDS], and 1× Complete Protease Inhibitor Cocktail). The protein lysates were mixed with SDS sample loading buffer containing β-mercaptoethanol, electrophoresed on a 15% SDS-polyacrylamide gel, and electrotransferred onto an Immobilon-P membrane (Millipore, Billerica, MA, USA). The membrane was blocked with PBS with Tween 20 (PBS-T) containing 5% Amersham^TM ECL^TM Blocking Agent (GE healthcare UK Ltd., Backinghamshire, England) overnight, then incubated with primary antibody, Mist1 antibody (Thermoscientific, Waltham, MA, USA), in blocking buffer for 2 hr. Then, the membrane was washed with PBS-T and incubated with a horseradish peroxidase-conjugated secondary antibody (Millipore). After another wash with PBS-T, immunoreactive protein was developed by the ECL reagent (Millipore) and the chemiluminescence was captured with an ATTO Ez-Capture MG AE-9300 (ATTO Corporation, Tokyo, Japan).