In vitro assay of the interaction between Rnc1 protein and Pmp1 mRNA by affinity capillary electrophoresis with a carboxylated capillary.
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<0.4%). Further, the proteins in human serum also gave sharp peaks and its repeatability was kept at a high level by pre-treatment of a capillary inner wall with 1M sodium chloride solution before each run. An Rnc1 protein was analyzed by ACE with background electrolytes containing various concentrations of Pmp1 sense mRNA using a carboxylated capillary. Increase in the concentration of the mRNA was found to delay the migration time of the protein. But the migration time of the protein was kept constant with increasing Pmp1 anti-sense mRNA instead of Pmp1 sense mRNA. A straight line (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 4.15x10(6)M(-1). These results suggest that the association constants of proteins with mRNAs as ligands were easily determined by the proposed method.
Taga
Atsushi
A
Faculty of Pharmacy, Kinki University, 3-4-1 Kowakae, Higashi-osaka 577-8502, Japan. punk@phar.kindai.ac.jp
Satoh
Ryosuke
R
Ishiwata
Shunji
S
Kodama
Shuji
S
Sato
Atsushi
A
Suzuki
Kentaro
K
Sugiura
Reiko
R
D000818:Animals; D002264:Carboxylic Acids / Q000737:chemistry / Q000378:metabolism*; D002417:Cattle; D003851:Deoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods; D006801:Humans; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding / Q000235:genetics; D011510:Proteolipids / Q000235:genetics / Q000378:metabolism*; D006180:Proton-Translocating ATPases / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D012359:tRNA Methyltransferases / Q000235:genetics / Q000378:metabolism*
D002264:Carboxylic Acids; D008565:Membrane Proteins; C489077:PMP1 protein, S cerevisiae; D011510:Proteolipids; D012333:RNA, Messenger; D029701:Saccharomyces cerevisiae Proteins; D012359:tRNA Methyltransferases; D003851:Deoxyribonucleases; C077501:TRM2 protein, S cerevisiae; D006180:Proton-Translocating ATPases