Biological Membranes as Protein Aggregation Matrices and Targets of Amyloid Toxicity - Protein Misfolding and Cellular Stress in Disease and Aging - page 227

interaction. The phase shift can be used to differentiate areas on
a sample with such differing properties as friction, adhesion, and
viscoelasticity. The technique is used simultaneously with DFM
mode, so that topography can be measured as well.
The samples for atomic force microscopy imaging should be
immobilized on a rigid support. Biological specimens like cells,
molecules (proteins), and Langmuir-Blodgett films are usually
bound to a flat substrate like mica or glass. Mica is a very common
support because the surface is:

atomically flat●●

●

clean after cleavage

●

easy to cut to desired sizes

●

relatively inexpensive

●

negatively charged but it can be modified to make the surface

●

positive

Sample preparation varies slightly depending on the protein itself.
Typically, protein concentration is in the 1-100 mg/ml range.
The incubation time also varies from as little as 15 min to up to
24 h. After proper incubation period, rinse the sample with fresh
buffer (see Note 8). For best results, use the narrow silicon nitride
tips for Tapping Mode AFM. It may also be necessary to adjust
the electrolyte solution and pH of the buffer in order to minimize
the force applied to the sample.

3.1.2. Sample Preparation

3.1.2.1. Sample
Preparation: Protein

1. Dissolve protein
Dissolve the protein in suitable buffer and at proper

●

concentration.
2. Prepare fluid cell
Prepare the fluid cell for Tapping Mode in fluid opera-

●

tion. Clean the fluid cell and load a probe. For best
results, clean the probe with UV light.
3. Prepare mica
Cleave a fresh mica surface by first pressing some adhe-

●

sive tape against the top mica surface, then peeling off the
tape. Glue mica to a small puck (e.g. using epoxy).
4. Deposit protein solution on mica
Deposit 50 mL of protein solution on the freshly cleaved

●

mica.
5. Allow protein to bind to substrate
Allow the protein solution to bind to the mica substrate

●

for 20-30 min. Binding time may vary with different
samples (it can be up to 24 h). For longer binding times,