title: Use of chromophoric ligands to visually screen co-crystals of putative protein-nucleic acid complexes.
abstract: Distinguishing between crystals of protein-nucleic acid complexes and those containing protein alone is a common problem in structural studies of protein-nucleic acid interactions. Currently, there are several methods available for detecting nucleic acid in crystals, including gel electrophoresis, SYBR Gold fluorescence dye staining, and methyl violet staining. However, they require either that the crystals be sacrificed or access to a fluorescence microscope. In this protocol, we describe an approach that allows direct visualization of either the presence or absence of oligonucleotides in crystals grown from solutions containing both protein and nucleic acid--labeling with the Cy5 dye. In addition to offering the advantage of being able to distinguish between crystals of complex and protein alone with the naked eye or a light microscope, crystals of covalently Cy5-labeled DNA can be directly used for X-ray diffraction data collection.
authors: Vanderbilt University School of Medicine, Nashville, Tennessee, USA.
authors: Xiaohua
authors: X
authors: Jiang
authors: Martin
authors: M
authors: Egli
mesh_terms: D002232:Carbocyanines / Q000737:chemistry; D003460:Crystallization; D017931:DNA Primers / Q000737:chemistry; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry; D004586:Electrophoresis / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry*; D005840:Gentian Violet / Q000737:chemistry; D008024:Ligands; D009696:Nucleic Acids / Q000737:chemistry* / Q000378:metabolism; D009841:Oligonucleotides / Q000737:chemistry; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D012996:Solutions; D000099270:Y-Family DNA Polymerases
chemical_list: D002232:Carbocyanines; D017931:DNA Primers; D004259:DNA-Directed DNA Polymerase; D005456:Fluorescent Dyes; D005840:Gentian Violet; D008024:Ligands; D009696:Nucleic Acids; D009841:Oligonucleotides; D011506:Proteins; D012996:Solutions; C085321:cyanine dye 5; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases