The purified GST protein and co-incubated peptides were maintained at 37°C in Eppendorf tubes and 1400 rpm for the duration of the experiment in an orbital mixer. At 24 h after cleavage of GST with factor Xa protease, a 5.0 μL aliquot of each protein incubation was spotted onto a freshly cleaved mica substrate (Ted Pella Inc., Redding, CA), for 30 seconds, washed with 200μl of ultra-pure water to remove salt and dried gently with a stream of clean air. The mica was placed on metal pucks and stored in a covered petri dish until imaging.
AFM imaging Experiments were performed with a Nanoscope V Multi-Mode scanning probe microscope (Veeco, Santa Barbara,CA) equipped with a closed loop vertical engage J-scanner. All images were acquired with diving board shaped silicon-oxide cantilevers with a nominal spring constant of 40 N/m and resonance frequency of about ~300 kHz. Scan rates were set at 1–2 Hz with cantilever drive frequencies 10% below resonance. All images were analyzed using Matlab with the image processing toolbox (MathWorks, Natick, MA) as previously described.18 (link)
AFM imaging Experiments were performed with a Nanoscope V Multi-Mode scanning probe microscope (Veeco, Santa Barbara,CA) equipped with a closed loop vertical engage J-scanner. All images were acquired with diving board shaped silicon-oxide cantilevers with a nominal spring constant of 40 N/m and resonance frequency of about ~300 kHz. Scan rates were set at 1–2 Hz with cantilever drive frequencies 10% below resonance. All images were analyzed using Matlab with the image processing toolbox (MathWorks, Natick, MA) as previously described.18 (link)
