Improvements in immunoprecipitation of specific messenger RNA. Isolation of highly purified conalbumin mRNA in high yield.
We have described previously procedures for the isolation of specific mRNA employing immunoprecipitation of polysomes. In spite of our success with ovalbumin mRNA in the chicken oviduct, we have had considerable difficulties in applying these same published techniques to the immunopurification of conalbumin mRNA, despite the fact that the chicken oviduct synthesizes up to 10% of protein as conalbumin. Here we describe a number of modifications and refinements which have proved essential in obtaining intact conalbumin mRNA in high purity and high yields. These refinements include: (a) improved purification of conalbumin in order to remove contaminating proteins that result in impure antibodies; (b) improved isolation of specific conalbumin antibody in high yields; (c) improved methods for reducing contamination by non-specific polysomes; (d) improved techniques for isolation of RNA from immunoprecipitates resulting in less degradation and higher recovery of conalbumin mRNA; (E) improved techniques for efficient translation of conalbumin mRNA involving treatment of the RNA with methylmercury prior to translation. We conclude that problems involved in the immunoprecipitation of different mRNAs may differ, and that various refinements in techniques may be required for obtaining highly purified preparations of intact mRNA in high yields.
D000818:Animals; D000937:Antigen-Antibody Reactions; D002458:Cell Fractionation / Q000379:methods; D002645:Chickens; D003207:Conalbumin / Q000096:biosynthesis*; D004527:Egg Proteins / Q000096:biosynthesis*; D005260:Female; D007700:Kinetics; D009693:Nucleic Acid Hybridization; D010057:Oviducts / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism / Q000648:ultrastructure*; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000302:isolation & purification*
D004527:Egg Proteins; D012333:RNA, Messenger; D003207:Conalbumin