Poly(ethylene oxide) facilitates the characterization of an affinity between strongly basic proteins with DNA by affinity capillary electrophoresis

Abstract

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.

Protein attachment onto silica surfaces – a survey of molecular fundamentals, resulting effects and novel preventive strategies in CE

“…If PEO of rather low M r (2 Â 10 5 ) was applied, daily regeneration of SiOH groups and a prewash sequence consisting of 1.0 mol/L HCl and an affiliated rinse with 0.2% PEO in 0.1 mol/L HCl became necessary between runs. This way, the EOF was reduced by 10% over 12 h and recovery of kin17 was 79%, which was probably related to the low ionic strength of the BGE [118].…”

“…Furthermore, preformed protein adsorbates can trigger a self-amplifying process, enhancing further protein aggregation and leading to irregular peak areas, pattern and t m [115]. The concept of Towns and Regnier was adapted to commercial single detector instruments, either by shortening the capillary after several runs [116,117] or by sample injection from the capillary inlet and outlet [118]. In either case the corrected peak areas attained from both capillary lengths were compared.…”

A novel PEG coating immobilized onto capillary through polydopamine coating for separation of proteins in CE

“…Capillaries coated with PEG-NH 2 polymer show almost the same strength EOF as the bare fused-silica capillary under the basic pH conditions. PEG adsorption form a stable coating via hydrogen bonding between the ether oxygen of PEG and the surface silanol groups of the capillary [25,26]. Especially, acidified PEG is necessary to form a stable coating [26,43].…”

“…Subsequently, the polydopaminecoated capillary was rinsed with water for 5 min to avoid residuary dopamine physically attached to the tube surface and then rinsed with the nitrogen stream for 10 min. After that, each polydopamine-coated capillary was filled with PEG-NH 2 solution with different concentrations (10,15,25, and 50 mg/mL PEG-NH 2 in 10 mM Tris-HCl buffer, pH 8.60), respectively and sealed on both ends with silicone. The sealed capillary was kept for another 30 h at 501C, then rinsed with water for 5 min and dried with the nitrogen stream for 15 min, and then polydopamine-graft-PEG coating capillary with different concentrations of PEG-NH 2 (including 10, 15, 25, and 50 mg/mL) was obtained.…”

“…One of the most extensively studied antifouling polymers is PEG and its grafted copolymers. They are usually water-soluble polymers with low toxicity and outstanding biocompatibility [24] and have widely been used to separate the proteins [12,13,19,25,26], polypeptide [27], and DNA [28] by CE. Although excellent separation efficiency could be obtained by using PEG-coated capillary, PEG is ineffective to prevent the protein adsorption after several consecutive runs because the PEG coating is easy to be washed away from the capillary wall due to its high hydrophilicity.…”