Journal
ANALYTICAL CHEMISTRY
Volume 94, Issue 5, Pages 2578-2588Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acs.analchem.1c04560
Keywords
-
Categories
Funding
- NSERC Canada [STPG-P 521331-2018]
Ask authors/readers for more resources
This study reveals the dependence of the partitioning of protein-DNA complexes on experimental conditions. By comparing two modes of capillary electrophoresis, NECEEM and IFCE, the optimal conditions for separating protein targets of different sizes can be determined. IFCE is preferable for large-size protein targets, while NECEEM should be the method of choice for small-size protein targets.
Partitioning of protein-DNA complexes from protein-unbound DNA is a key step in selection of DNA aptamers. Conceptually, the partitioning step is characterized by two parameters: transmittance for protein-bound DNA (binders) and transmittance for unbound DNA (nonbinders). Here, we present the first study to reveal how these transmittances depend on experimental conditions; such studies are pivotal to the effective planning and control of selection. Our focus was capillary electrophoresis (CE), which is a partitioning approach of high efficiency. By combining a theoretical model and experimental data, we evaluated the dependence of transmittances of binders and nonbinders on the molecular weight of the protein target in two modes of CE-based partitioning: nonequilibrium capillary electrophoresis of equilibrium mixtures (NECEEM) and ideal-filter capillary electrophoresis (IFCE). Our data suggest that as the molecular weight of the protein target decreases: (i) the transmittance for binders remains close to unity in NECEEM but decreases drastically in IFCE and (ii) the transmittance for nonbinders increases orders of magnitude in NECEEM but remains relatively stable at a very low level in IFCE. To determine the optimal CE conditions for a given size of protein target, a balance between transmittances of binders and nonbinders must be reached; such a balance would ensure the collection of binders of sufficient purity and quantity. We conclude that, as a rule of thumb, IFCE is preferable for large-size protein targets while NECEEM should be the method of choice for small-size protein targets.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available