Cumbers:protocols/Invitrogen NuPage gels

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Phosphorylated epitope?

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.

4H8 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.