Afm integra vita

Stored solutions of purified Dps were passed through a Sephadex G-15 column (1x5 cm³) to remove any aggregated particles. Collected fractions were diluted in the buffer containing 50 mM Tris-HCl (pH 7.5) and 10 mM NaCl, to the final concentration 1 ng/μl (4.4 nM of dodecamers) and 2 μl of this solution were deposited on mica for scanning. Linear DNA fragments or Y-shape structures were dissolved in 5 mM MgCl₂ to the concentration of 1 ng/μl (4–19 nM). The Dps complexes with different DNA fragments were formed at room temperature in the buffer: 50 mM Tris-HCl (pH 7.5), 10 mM NaCl and 5 mM MgCl₂ (10 μl) for 30 minutes and loaded on mica. Three-ten molar excess of linear DNA fragments or five-fifty molar excess of branched molecules was used for complex formation. Control samples were prepared by the same way, but without Dps. All samples were hold on mica for 5 minutes, washed twice with water for 30 seconds, dried, and the structure of complexes formed was analyzed by AFM Integra-Vita (NT-MDT, Russia) using cantilevers NSG03 with 10 nm tip curvature radius and 47–150 kHz resonance frequency. Measurements were done in semi-contact (tapping) mode. Images obtained were analyzed by Nova software (NT-MDT, Russia).

Sample preparation for atomic force microscopy was performed as described in [18 (link)]. This included filtration of protein samples through Sephadex G-15 column (1 × 5 cms) and dilution in the buffer containing 50 mM Tris-HCl (pH 7.5) with 10 mM NaCl, to the final concentration of 1 ng/μL (<4.4 nM of dodecamers). Two microliters of this solution were loaded on mica and held there for 5 min. Then, the sample was washed, dried, and scanned by AFM Integra-Vita (NT-MDT, Zelenograd Russia).