I am using 2D gel electrophoresis to visualize polyubiquitinated proteins. However, while I can see actin and heat shock protein using when appropriate antibodies, I cannot visualize them using anti-ubiquitin antibody. Could it be degradation problem?
I run isoelectric focusing at 200 V overnight (at least 18 hours) and 700 V the last 30 minutes. I am worried that I am applying more volts than needed, because the standard protocol is 200 V for 20 minutes, 450 V for 15 minutes, 750 V for 15 minutes, 2000 V for 30 minutes, but due to problems with the power unit I cannot do this.
I am sure I have polyubiquitination because I use a drug that triggers polyubiquitinated protein accumulation inside the cells.
I would like to add also that cells are collected by scraping in cold PBS and then they are placed in ice and the pellet is also collected at 4 degrees to prevent protein degradation.