Western blotting was carried out according to standard procedures. Transfer conditions were 100 V for 3 hours at 4°C in 10% methanol for experiments involving detection of high–molecular weight proteins and were 330 mA for 1 hour and 30 min in 20% methanol for routine transfers. Membranes were blocked in 5% milk/TBST (tris buffered saline with 0.1% Tween20) before overnight incubation at 4°C with primary antibodies at a 1:1000 dilution. The following primary antibodies were used: mouse anti–c-Myc (A7; sc-56634, Santa Cruz Biotechnology), mouse anti-Flag (M2; F3165, Sigma-Aldrich), mouse anti-HA (901502, BioLegend), rabbit anti-HA (3724, Cell Signaling Technology), mouse anti-HDAC2 (05-814, Merck Millipore), mouse anti-HSP90 (Ab13492, Abcam), rabbit anti-RBBP4 (79416, Abcam), rabbit-RBBP7 (R4279, Sigma-Aldrich), goat anti-HSP90 (sc-1055, Santa Cruz Biotechnology), rabbit anti-CREB (9197, Cell Signaling Technology), and phospho-CREB ser133 (9191S, Cell Signaling Technology). After TBST washes, membranes were incubated with the appropriate secondary antibody at a 1:20,000 dilution. The following secondary antibodies were used: anti-mouse horseradish peroxidase (HRP; NXA931, GE Healthcare), anti-goat HRP (A5420, Sigma-Aldrich), and anti-rabbit HRP (NA93AV, GE Healthcare). Signal was detected by using enhanced chemiluminesence (ECL) or ECL Prime detecting reagents (GE Healthcare Life Sciences) and by exposing the immunoblot to the Amersham Hyperfilm (GE Healthcare Life Sciences).

Note: The content above has been extracted from a research article, so it may not display correctly.

Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.

We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.