Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction
Published: June 29, 2023
Abstract
Source: Mittermaier, A. K., et al. Measuring Biomolecular DSC Profiles with Thermolabile Ligands to Rapidly Characterize Folding and Binding Interactions. J. Vis. Exp. (2017)
This video demonstrates differential scanning calorimetry — a technique to study the interaction between a biomolecule and a ligand. The binding of a DNA aptamer and a thermolabile ligand is studied by applying repeated heating and cooling cycles to the sample and obtaining scans for thermodynamic analysis.
Protocol
1. Sample Preparation Purify the desired biomolecule. NOTE: This protocol uses purchased cocaine-binding DNA aptamer MN4 after exchanging against 2 M NaCl three times followed by three rounds of deionized water using a centrifugal filter with a 3 kDa molecular weight cut-off membrane. Synthesize and purify, or purchase the desired thermolabile ligand. NOTE: MN4 binds the thermolabile ligand cocaine. MN4 also binds quinine, which is use…
Açıklamalar
The authors have nothing to disclose.
Materials
| Sodium chloride | Chem Impex | #00829 | |
| Sodium phosphate monobasic dihydrate | Sigma Aldrich | 71502 | |
| Sodium phosphate dibasic | Sigma Aldrich | S9763 | |
| Deioinized water for molecular biology | Millipore | H20MB1001 | |
| 0.2-micron sterile syringe filters | VWR | CA28145-477 | |
| 3 kDa centrifugal filters | Millipore | UFC900324 | |
| Dialysis tubing 0.5-1.0 kDa cutoff | Spectrum Laboratories | 131048 | |
| Silicon tubing | VWR | 89068-474 | |
| Plastic DSC flange caps | TA Instruments | 6111 | |
| DNA aptamer MN4 | Integrated DNA Technologies | https://www.idtdna.com/site/order/menu | |
| Cocaine | Sigma Aldrich | C008 | |
| Quinine | Sigma Aldrich | 22620 | |
| NanoDSC-III microcalorimeter | TA Instruments | http://www.tainstruments.com/nanodsc/ | |
| DSCRun software | TA Instruments | http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/ | |
| NanoAnalyze software | TA Instruments | http://www.tainstruments.com/support/software-downloads-support/instruments-by-software/ | |
| Contrad-70 | VWR | 89233-152 |
Etiketler
Bu Makaleden Alıntı Yapın
Differential Scanning Calorimetry to Study DNA Aptamer-Thermolabile Ligand Interaction. J. Vis. Exp. (Pending Publication), e21450, doi: (2023).