Selection of surfactants for cell lysis in chemical cytometry to study protein-DNA interactions.
Protein-DNA interactions play a defining role in many cellular processes. Studying such interactions at the single-cell level is important and challenging. Here we make the first step toward achieving this goal with chemical cytometry. Chemical cytometry utilizes capillary separation for detailed chemical analyses of single cells. The cell is injected into a capillary, lysed, and its components are analyzed by CE or capillary chromatography with highly sensitive detection. In order to apply chemical cytometry to studies of protein-DNA interactions, cell lysis must not destroy protein-DNA complexes. Surfactants represent the most practical means of cell lysis inside the capillary. This work aimed at finding surfactants and lysis conditions that do not destroy protein-DNA complexes. We studied three groups of surfactants--ionic, zwitterionic, and nonionic--with respect to their ability to lyse the cell membrane without significantly influencing the stability of protein-DNA complexes. Nonequilibrium CE of equilibrium mixtures with surfactants in the equilibrium mixtures and in the run buffer was used to measure the equilibrium constant, K(d), and rate constant, k(off), of protein-DNA complex dissociation. We found that nonionic surfactants worked best: they lyse the plasma membrane without significantly influencing K(d), k(off), or the EOF. This work creates the foundation for studies of protein-DNA interactions in single cells by chemical cytometry.
Department of Chemistry, York University, Toronto, Ontario, Canada.
Zhulin
Z
Pang
Azza
A
Al-Mahrouki
Maxim
M
Berezovski
Sergey N
SN
Krylov
D002469:Cell Separation / Q000379:methods*; D002477:Cells / Q000187:drug effects*; D003592:Cytophotometry / Q000379:methods*; D004247:DNA / Q000378:metabolism*; D019075:Electrophoresis, Capillary / Q000379:methods; D025941:Protein Interaction Mapping / Q000379:methods*; D013501:Surface-Active Agents / Q000494:pharmacology*
D013501:Surface-Active Agents; D004247:DNA