Poly(ethylene oxide) facilitates the characterization of an affinity between strongly basic proteins with DNA by affinity capillary electrophoresis.
In order to study kin17 protein-DNA affinity, we have developed a fast and reproducible capillary electrophoresis (CE) analysis of a strongly basic protein: kin17 protein, using a nonpermanent coating based on poly(ethylene oxide) (PEO) to avoid adsorption of kin17. The coating procedure was optimized to provide a residual and stable electroosmotic flow (EOF = 5 x 10(-5) cm(2)/V x s), exhibiting RSD of 0.3% and excellent long-term stability. Good intraday and interday reproducibility of kin17 migration times (0.8 and 0.3% relative standard deviation (RSD), respectively) enabled us to consider that the recovery percentage obtained for kin17 protein was satisfactory (79%). The potential of this PEO-based coating procedure was evaluated for affinity CE method in order to study the affinity of kin17 protein for two single-stranded DNA (ssDNA) models: polydeoxyadenylic acid and polydeoxycytidilic acid (pdA and pdC). Binding constants (1.5 x 10(7) +/- 17% and 1.7 x 10(7) + 25%M(-1)) were evaluated assuming a 1:1 affinity between kin17 and pdA or pdC, respectively.
Tran
Nguyet Thuy
NT
Groupe de Chimie Analytique Paris-Sud, EA 3343, Faculté de Pharmacie, Université Paris XI, F-92290 Châtenay-Malabry, France. thuy.tran@cep.u-psud.fr
Taverna
Myriam
M
Miccoli
Laurent
L
Angulo
Jaime F
JF
D000180:Acrylic Resins / Q000737:chemistry; D000327:Adsorption; D004247:DNA / Q000302:isolation & purification*; D004268:DNA-Binding Proteins / Q000302:isolation & purification*; D019075:Electrophoresis, Capillary / Q000295:instrumentation / Q000379:methods*; D009687:Nuclear Proteins / Q000302:isolation & purification*; D009995:Osmosis; D011061:Poly A / Q000737:chemistry; D011066:Poly C / Q000737:chemistry; D011089:Polydeoxyribonucleotides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry*; D011485:Protein Binding; D016601:RNA-Binding Proteins; D000099166:Polyacrylamides
D000180:Acrylic Resins; D004247:DNA; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D011061:Poly A; D011066:Poly C; D011089:Polydeoxyribonucleotides; D011092:Polyethylene Glycols; D016601:RNA-Binding Proteins; D000099166:Polyacrylamides; C489487:KIN protein, human; C015465:poly(dA); C023826:poly(dC)