In vitro assay of the interaction between Rnc1 protein and Pmp1 mRNA by affinity capillary electrophoresis with a carboxylated capillary

Abstract The interaction between Rnc1, an RNA interactive protein, and a Pmp1 mRNA was investigated by affinity capillary electrophoresis (ACE). Prior to the ACE experiments, the column performances of three capillaries (an untreated fused silica capillary, a polybrene–polyacrylic acid (PB–PAA) double layer coating capillary, and a carboxylated capillary with a covalent modification) were studied with model proteins including ribonuclease B (RNase B) and bovine serum albumin (BSA). Using an untreated fused silica and a PB–PAA double layer coating capillaries, both of the protein peaks were broad and tailing. However, using a carboxylated capillary, the protein peaks were sharp and symmetric, and migration times were repeatable (RSD  r  = 0.987) was obtained by plotting 1/(migration time shift) versus 1/(Pmp1 sense mRNA concentration) and the association constant of Rnc1 protein with Pmp1 sense mRNA could be estimated to be 4.15 × 10 6  M −1 . These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method.