Western Blotting of Protein Expression

Western blots were performed using standard lab procedures. Log phase cultures were lysed in SDS-PAGE loading buffer and boiled for 10 minutes. Equivalent OD units of cell lysate were loaded. Standard procedures were followed for SDS-PAGE and protein transfer to nitrocellulose membrane. Antibodies were used at the following concentrations: Primary antibodies used were Flag-M2 – 1:1,000 (Sigma, St. Louis, Missouri) and CdnL −1:2,500 (33 (link)). Secondary antibodies used were 1:10,000 of HRP-labeled α-mouse (for Flag) (PerkinElmer) or α-rabbit (PerkinElmer) (for CdnL). Chemiluminescent substrate (PerkinElmer) was added to visualize proteins via an Amersham Imager 600 RGB gel and membrane imager (GE).