A sensitive two-color electrophoretic mobility shift assay for detecting both nucleic acids and protein in gels.
DNA-binding proteins are key to the regulation and control of gene expression, replication and recombination. The electrophoretic mobility shift assay (or gel shift assay) is considered an essential tool in modern molecular biology for the study of protein-nucleic acid interactions. As typically implemented, however, the technique suffers from a number of shortcomings, including the handling of hazardous (32)P-labeled DNA probes, and difficulty in quantifying the amount of DNA and especially the amount of protein in the gel. A new detection method for mobility-shift assays is described that represents a significant improvement over existing techniques. The assay is fast, simple, does not require the use of radioisotopes and allows independent quantitative determination of: (i) free nucleic acid, (ii) bound nucleic acid, (iii) bound protein, and (iv) free protein. Nucleic acids are detected with SYBR Green EMSA dye, while proteins are subsequently detected with SYPRO Ruby EMSA dye. All fluorescence staining steps are performed after the entire gel-shift experiment is completed, so there is no need to prelabel either the DNA or the protein and no possibility of the fluorescent reagents interfering with the protein-nucleic acid interactions. The ability to independently quantify each molecular species allows more rigorous data analysis methods to be applied, especially with respect to the mass of protein bound per nucleic acid.
Jing
Debra
D
Proteomics Section, Molecular Probes Inc., 29851 Willow Creek Road, Eugene, OR 97402, USA. debra.jing@probes.com
Agnew
Jakyoung
J
Patton
Wayne F
WF
Hendrickson
Jill
J
Beechem
Joseph M
JM
D004247:DNA / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000494:pharmacology; D007700:Kinetics; D008856:Microscopy, Fluorescence; D009696:Nucleic Acids / Q000737:chemistry*; D009841:Oligonucleotides / Q000737:chemistry; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry
D004268:DNA-Binding Proteins; D005456:Fluorescent Dyes; D009696:Nucleic Acids; D009841:Oligonucleotides; D011506:Proteins; D004247:DNA