Instruction manual

Fluid Operation
Lysozyme on Mica—A Model Procedure for Protein Binding
Rev. B MultiMode SPM Instruction Manual 143
• TappingMode Fluid Cell, Model MMTFC
• Cantilevers (Oxide-Sharpened Silicon Nitride tips, Model NP-S, work well)
• Source of filtered (0.2 µm), compressed air or dry nitrogen
• UV lamp, high-intensity; Oriel Mod. 6035 pencil-style spectral calibration lamp or
equivalent (optional for cantilever cleaning).
• Syringes: (1) 1 cc; (2) 5 cc; Micropipettes
• Fluid cell liquid lines (silicone tubing and fittings)
• Fluid cell o-ring (optional, see Method 1 (with an O-ring): Page 127).
• Hemostats or similar clamping device (for liquid lines).
• Filter paper
2. Dissolve the lysozyme in phosphate buffer (PBS) solution to a concentration of 1µg per ml
(this concentration provides a convenient coverage for AFM imaging and may be used for a
variety of similar size samples). This mixture should be drawn into a clean, 1 cc syringe and
capped. Prepare another 5 cc syringe of straight buffer solution.
3. Prepare the fluid cell for TappingMode in fluid operation. Clean the fluid cell and load a
cantilever. For best results, clean the cantilever with UV light.
4. Cleave a fresh mica surface by first pressing some adhesive tape against the top mica
surfaces, then peeling off the tape.
5. Deposit 50µl of protein solution on the freshly cleaved mica.
6. Allow 20-30 minutes for the protein solution to bind to the mica substrate. Binding time may
vary with different samples. For longer binding times, put the mica in a covered dish with a
wet piece of filter paper to keep the liquid from evaporating.
7. Rinse the sample with a large quantity of buffer to remove unbound protein. Leave a drop of
buffer on the mica.
8. Mount the sample on the scanner end cap. Seal the fluid cell and fill with buffer.
CAUTION: When imaging fluid samples, use extraordinary precautions against
spillage.