After SDS-PAGE separation of proteins, transfer sandwich was prepared with PVDF membrane and placed in transfer buffer. PVDF membrane (Amersham) was activated with methanol for 15 s prior to use. The blot was allowed to run for 120 min at 100 volts. After the transfer of proteins, membrane was blocked using 5% skimmed milk powder as blocking buffer for 1 h at RT. Then membrane was washed with 1× TBST buffer and incubated with primary antibody overnight (details of antibody in Supplementary Table 3 ). Again, membrane was washed in 1× TBST buffer followed by secondary antibody (1:5,000 dilution) treatment, then incubated for 1 h at RT. After incubation, membrane was washed with 1× TBST buffer three times and detected with ECL reagent (TAKARA, Japan) using Versa Doc (BD Bioscience, USA) or X-ray film.
Ecl reagent
Manufactured by Takara Bio
Sourced in Japan, United Kingdom
ECL reagent is a chemiluminescent detection solution used for Western blotting analysis. It is designed to detect and visualize target proteins labeled with horseradish peroxidase (HRP) conjugates.
Automatically generated - may contain errors
Lab products found in correlation
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (2 mentions)
Pvdf membrane, by Cytiva (1 mentions)
Amersham ecltm blocking agent, by GE Healthcare (1 mentions)
Immobilon p membrane, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Merck Group (1 mentions)
Imagequant las 500, by GE Healthcare (1 mentions)
2 protocols using ecl reagent
1
Western Blot Protocol for Protein Analysis
2
Nuclear Extraction and Western Blot Analysis
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Nuclear extracts were prepared from livers or hepatocytes using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Thermo Fisher Scientific) according to the manufacture's protocol. Nuclear extracts were mixed with SDS sample loading buffer (Wako Pure Chemical Corporation, Osaka, Japan), electrophoresed using a Super-Sep TM Ace 10% gel (Wako Pure Chemical Corporation), and then electrotransferred onto an Immobilon-P membrane (Millipore, Billerica, MA). The membrane was blocked overnight in phosphate buffered saline (PBS) with Tween 20 (PBS-T) containing 3% or 5% Amersham TM ECL TM Blocking Agent (GE healthcare UK Ltd.) and then incubated with primary antibody-Bcl6 antibody (D-8; Santa Cruz Biotechnology, Dallas, TX)-for 2 h. Next, the membrane was washed with PBS-T and incubated with horseradish peroxidase-conjugated secondary antibody (Millipore). During incubation with primary and secondary antibodies, western blot immune booster solutions (Takara Bio Inc., Shiga, Japan) were used to enhance antibody interactions. After another wash with PBS-T, immunoreactive proteins were developed by the ECL reagent (Takara Bio Inc.) and chemiluminescence was captured using the Image Quant LAS 500 (GE healthcare UK Ltd.).
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