Electrophoretic Mobility Shift Assays using Infrared-Fluorescent DNA Probes v1

Abstract

Protein-DNA binding interactions are critical in several biological processes,especiallythe regulation of gene expression at the level of transcription initiation. An important technique for studyingthese interactions is the electrophoretic mobility shift assay (EMSA), whereby protein-DNA complexes are resolved on the basis of their mass:charge ratio using native polyacrylamidegel electrophoresis (nPAGE).Here we describe EMSA using PCR-generated, near infrared-fluorescent DNA probes,and IR fluorescence imaging to qualitatively and quantitatively study the interaction of transcriptional regulatory proteins fromthermophilic organisms with different DNAs. Direct imaging of IR fluorophore-labeled DNA probes is advantageous because itprovides high sensitivity (subnanomolar) without the need for intermediate staining steps or costly and problematic radiolabeled probes,thereby providing a more affordable and sensitive option to image protein-DNA on polyacrylamide gels by techniques such as EMSA.

Identification and Characterization of Preferred DNA-Binding Sites for the Thermus thermophilus HB8 Transcriptional Regulator TTHA0973

“…Electrophoretic mobility shift assays (EMSA) with both libraries and defined DNAs were performed essentially as previously described [7,8], with a detailed protocol being available [19]. Biolayer interferometry was performed essentially as previously described [7], with the exception that only four concentrations of TTHA0973 (11, 33, 100, 300 nM) were used for each DNA probe investigated.…”

General and Genomic DNA-Binding Specificity for the Thermus thermophilus HB8 Transcription Factor TTHB023

“…Following 5 min cooling to 37 • C, 0.2 U IISRE was added, and incubations continued for an additional 5 min before stopping on ice. DNA cleavage was analyzed by native PAGE and IR fluorescence imaging, as described [24]. Comparisons were made between reactions without IISRE, without TTHB023, and with both being present (see Figure 1, Lanes 1, 2, and 3, respectively) to determine if ligand-dependent, cleavage resistance were present.…”

“…Electrophoretic mobility shift assays (EMSAs) with both Round 6 pool and TTHB023 consensus DNA were performed as previously described [4,5], with a detailed protocol being available [24]. Binding reactions (5 µL) consisted of 1 ng (21 fmoles) DNA and indicted concentration of TTHB023 in 1 × NEB CutSmart buffer.…”

“…Binding reactions (5 µL) consisted of 1 ng (21 fmoles) DNA and indicted concentration of TTHB023 in 1 × NEB CutSmart buffer. After incubation (20 min, 55 • C), 2 µL 20% glucose + 0.9% Orange G was added and samples analyzed by 10% native PAGE, as described [4,24]. Protein-DNA complexes and free DNA were identified and quantitated using IR fluorescence imaging.…”

“…Apparent dissociation constants were then determined using data from the binding midpoint (approx. 50:50 complex:free DNA) and a standard binding equilibrium equation, as previously [4,24].…”