For Westerns: If you are using an antibody that is specific to a phosphorylated epitope, DO NOT use milk to block. Kinases in the milk will nonspecifically phosphorylate proteins in your sample. Use BSA as a blocking agent. It is important to block at least overnight. BSA is not as good of a blocking agent and you will see more background.
From Brodsky Genome Biology Paper
- 8WG16 recognize the hypophosphorylated (PolIIa)
- 4H8, a phosphorylation-independent state of the CTD of polymerase II (PolII)
- Thus, the 4H8 antibody is recognizing the total polymerase II population.
http://www.abcam.com/index.html?datasheet=5408 RNA polymerase II carboxy-terminal domain (CTD) interacts with a large multisubunit complex that contains TATA-binding protein (TBP) and is an integral part of the transcription initiation complex. Phosphorylation of RNA polymerase II's largest subunit C-terminal domain (CTD) is a key event during mRNA metabolism. Numerous enzymes, including cell cycle-dependent kinases and TFIIF-dependent phosphatases target the CTD.
- ChIP: Use 1-4µg per 25µg of chromatin.