Protein-based Biointerfaces to Control Stem Cell Differentiation - Biointerfaces: Where Material Meets Biology - page 18

NOTE: In such situations it will not be possible to evaluate effects of the
surface topography on proteins conformation.

d n 3 r 4 n g | 1

TIP: If a silicon surface is not the preferred substrate and polishing is not
trivial, layers of different materials of interest can be easily deposited on flat
substrate. For examples to test embolization of protein to polymers such as
polystyrene and PLA they can be spin coated on silicon wafer. This will pro-
vide flat surface that can allow distinguishing features below 1 nm.

A protocol to visualize proteins on a surface is as follows:

1. Prepare a flat substrate with the roughness below the smallest protein
(biomolecule) you plan to visualize.

TIP: Typically, a silicon wafer offers the desired roughness/flatness.

2. Modify the surface as required.
3. Scan the surface using atomic force microscopy. (For AFM settings, see
below.)
4. Immobilize the protein of interest. Typically, immobilization is carried
out by immersing the sample in the solution that contains protein.
5. Rinse the sample with ultra-pure water or phosphate-buffered saline
(PBS) to remove unbound and loosely attached proteins.
6. Scan the surface after immobilization.

.

NOTE: Scanning can be done both in air and in liquid. When you scan
the surface in air the surface must be dry.

7. Present the results. Typically, the height (topography) of the image both
in 2D and 3D are used to present the proteins present on the surface. In
addition, line profiles can be used to quantify the amount of proteins
(Figure 1.3).

The scanning settings are:

Scan mode: AC (tapping, non-contact mode); probe with a spring con-
stant 40 N m 1 (a softer tip will also work; occasionally depending on
the protein length softer probes might be required). Free amplitude of
the tip can be in the few hundred nanometers to 1 mm.

TIP: If the proteins are not well presented on the surface, the drive
amplitude can be decreased, followed by a decrease of the set point. This
will reduce the amplitude and should result in sharper images of proteins.