This paper describes procedures for studies of DNA and protein-DNA complexes by using atomic force microscopy (AFM) techniques. The necessary specifics for the preparation of functionalized mica surfaces with the use of (3-Aminopropyl) triethoxysilane (APTES) are provided.
In recent years, (AFM)-based methods to image DNA have grown increasingly popular. AFM has emerged as the perfect tool for the study of molecules at the nanoscale level. Numerous developments of AFM methods play a crucial role in the invention of new drugs, their delivery systems, and in the examination of disease-related tissue changes. Atomic force microscopy has also become a powerful tool due to its ability to scan a surface with nanometer resolution in aqueous liquids. This allows us to analyze biomolecules at the single molecular level.
In the Xu lab, AP-Mica was prepared by functionalizing mica sheets with APTES. Once functionalized, samples could be deposited onto the AP-Mica and an atomic force microscope was used to produce images of DNA-protein complexes. These images could be analyzed to understand DNA-protein interactions and their binding sites.
Preparation of AP-Mica
In order to analyze compounds through AFM, mica sheets had to be functionalized in order to bind DNA and proteins. The steps taken to functionalize mica sheets were as follows:
1. A plastic cap was placed on the bottom of a desiccator.
2. The desiccator was vacuumed to create an airtight seal then filled with nitrogen.
3. Mica sheets were cut into 5 cm x 5 cm dimensions and cleaved into ~0.1mm thickness.
4. 30uL APTES was placed into the plastic cap that was at the bottom of the desiccator.
5. Mounted mica sheets at top of desiccator.
6. Closed lid and allowed the functionalization reaction to proceed for 2 hours at room temperature.
7. After 2 hours, removed the APTES and purged the desiccator with nitrogen for 2 minutes.
8. The mica sheets were left in the desiccator for 1-2 days to cure. After cured, samples could be deposited onto the AP-Mica for analysis.
Figure 1: This figure shows a similar setup that was used in the Xu lab. Arrows 1 and 2 are plastic caps, arrow 3 is the mica sheet, and arrow 4 is the rod that the mica sheet was clipped to.
AFM Sample Analysis
After the AP-Mica is prepared, samples of DNA and DNA-protein complexes can be deposited and analyzed through AFM. The sample prepared...