Effect of buffer, electric field, and separation time on detection of aptamer-ligand complexes for affinity probe capillary electrophoresis.
The separation and detection of complexes of aptamers and protein targets by capillary electrophoresis (CE) with laser-induced fluorescence was examined. Aptamer-thrombin and aptamer-immunoglobulin E (IgE) were used as model systems. Phosphate, 3-(N-morpholino)propanesulfonic acid with phosphate, and tris(hydroxyamino)methane-glycine-potassium (TGK) buffer at pH 8.4 were tested as electrophoresis media. Buffer had a large effect with TGK providing the most stable complexes for both protein-aptamer complexes. Conditions that suppressed electroosmotic flow, such as addition of hydroxypropylmethylcellulose to the media or modification of the capillary inner wall with polyacrylamide, were found to prevent detection of complexes. The effect of separation time and electric field were evaluated by monitoring complexes with electric field varied from 150-2850 V/cm and effective column lengths of 3.5 and 7.0 cm. As expected, shorter times on the column greatly increased peak heights for the complexes due to a combination of less dilution by diffusion and less dissociation on the column. High fields were found to have a detrimental effect on detection of complexes. It is concluded that the best conditions for detection of noncovalent complexes involve use of the minimal column length and electric field necessary to achieve separation. The results will be of interest in developing affinity probe CE assays wherein aptamers are used as affinity ligands.
Buchanan
Danielle D
DD
Department of Chemistry, University of Florida, Gainesville, FL, USA.
Jameson
Emily E
EE
Perlette
Jon
J
Malik
Abdul
A
Kennedy
Robert T
RT
D000818:Animals; D002021:Buffers; D002846:Chromatography, Affinity / Q000379:methods*; D004277:DNA, Single-Stranded; D019075:Electrophoresis, Capillary / Q000379:methods*; D006801:Humans; D007073:Immunoglobulin E / Q000302:isolation & purification; D008024:Ligands; D009841:Oligonucleotides / Q000737:chemistry*; D011506:Proteins / Q000302:isolation & purification*; D012212:Rheology; D055672:Static Electricity; D013917:Thrombin / Q000302:isolation & purification; D013997:Time Factors
D002021:Buffers; D004277:DNA, Single-Stranded; D008024:Ligands; D009841:Oligonucleotides; D011506:Proteins; D007073:Immunoglobulin E; D013917:Thrombin