pre-immune serum in CHIP IP - (Jun/26/2006 )
I am just curious about one step described in one of CHIP protocols. In this protocol, they preclear chromatin by incubaing ~1 mL soluble chromatin with 2 ug sheared salmon sperm DNA, 20 µl pre-immune serum and protein-A sepharose slurry for 2 hr. a 4C. I do know that the sheared salmon sperm DNA is used to prevent non-specific of DNA binding to beads. However, I do not understand why they add pre-immune serum to the sepharose slurry? Does anybody know the purpose of adding the preimmune serum for preclearing the chromatin? Also, should they add sheared salmon sperm DNA to the beads, incubate for a while, and then perform preclearing step in order to block the beads?
I think this is a strange preclearing. I use salmon sperm and BSA (one can use preimmune serum as well, I think) to block the protein A. I preclear then the chromatin with a part of this prot.A and then I go on with the protocol. Also, I prefer to use Staphilococcus aureus cells, coated with protein A, because I feel more confident I'm pipeting exactly the same amount for each sample.