Pointprobe ppp fmr probes

TRF2 (80 nM dimer) without or with TIN2 (TIN2S or TIN2L, 80, 120 and 200 nM) was incubated with the linear pT270 DNA (10.5 nM) in TRF2 Imaging Buffer (20 mM HEPES [pH 7.5], 100 mM NaCl) at room temperature for 10 min. All DNA–protein samples were diluted 10-fold in TRF2 Imaging Buffer before being deposited on to a freshly prepared 1-(3-aminopropyl)silatrane (APS)-treated mica (SPI Supply) surface for 30 s (56 (link)). The APS-treated mica surface was washed with DI water and dried under a stream of nitrogen gas. All images were collected in the tapping mode using Pointprobe® PPP-FMR probes (Nanosensors, spring constants at ∼2.8 N/m) on an MFP-3D-Bio AFM (Asylum Research). All images were captured at a scan rate of 1–2 Hz, a scan size of 1–3 μm × 1–3 μm and a resolution of 512 × 512 pixels.

All DNA and protein samples were diluted in 1× AFM buffer [25 mM HEPES–KOH (pH 7.5), 25 mM NaOAc and 10 mM Mg(OAc)₂] before being deposited onto freshly cleaved mica surface (SPI Supply). Then, the samples were washed with MilliQ water and dried under a stream of nitrogen gas. The final protein, QD and DNA concentrations were 6.7 nM, 6.7 nM and 0.58 μg/ml, respectively. When QDs were not included, the final concentrations of SA1 and TRF1 proteins were 30 and 50 nM, respectively. All images were collected in the AC mode using a MFP-3D-Bio AFM (Asylum Research). Pointprobe^® PPP-FMR probes (Nanosensors) with spring constants at ∼2.8 N/m (nominal value) were used. All images were captured at a scan size of 1–3 × 1–3 μm, a scan rate of 1–2 Hz and a resolution of 512 × 512 pixels. The positions of proteins and protein-QDs on DNA were analyzed using the software from Asylum Research.