312 Questions
TIRF evanescent excitation field is D nm, generate an approximate expression for
the SNR of the spots using such TIRF excitation.
b
Explain under what conditions it might be suitable to use TIRF to monitor gene
expression of these proteins.
7.7
If two crystal structures, A and B, for a given protein molecule M are possible that
both have roughly the same interfacial energies and saturation values, but structure B
has a more open conformation such that the mean volume per molecule in the crystal
is greater than that in structure A by a factor of ~2, explain with reasoning what rela
tive % proportion by number of crystals one might expect when crystals are grown
spontaneously from a purified supersaturated solution of M.
7.8
Platinum wire of length 20 cm and diameter 75 μm was wound around a tungsten
electrical resistance heating filament that was then heated to evaporate all of the plat
inum 3 cm away from the surface of a glass coverslip surface of size 22 × 22 mm
under high vacuum to coat the surface in a thin layer of platinum for manufacturing
an optical filter. The coated coverslip was used in an inverted fluorescence micro
scope flow cell for detecting a specific protein that was labeled with a single fluores
cent dye molecule, conjugated to the coated coverslip surface. If the brightness of a
single fluorescently labeled protein on an identical coverslip not coated in platinum
was measured at ~7600 counts under the same imaging conditions, estimate with
reasoning what range of spot brightness values you might observe for the case of
using the platinum-coated coverslip. (Assume that the optical attenuation is roughly
linear with thickness of the platinum layer, equivalent to ~15% at 50 nm thickness,
and wavelength is independent across the visible light spectrum.)
7.9
A leak-free horizontal microfluidics device of length 15 mm was made consisting of
three cylindrical pipes each of length 5 mm with increasing diameters of 10, 20, and
30 μm attached end to end. If the flow was gravity driven due to a reservoir of fluid
placed 50 cm above the entrance of the flow cell connected via low-friction tubing,
estimate the time it takes for a 1 μm bead to flow from one end of the flow cell to
the other.
7.10 A competing flow-cell design to Question 7.9 was used, which consisted of three
pipes of the same diameters as the previous but each of length 15 mm connected this
time in parallel. How long will a similar bead take to flow from one end of the flow
cell to the other?
7.11 High-throughput methods for measuring protein–protein interaction kinetics using
cell lysate analysis in vitro can generate large errors compared to the in vivo kin
etics. Give a specific example of a protein–protein interaction whose kinetics can be
measured inside a living cell using a biophysical technique. What are the challenges to
using such assays for systems biology? Suggest a design of apparatus that might pro
vide high-throughput measurement and automated detection?
7.12 In internal radiotherapy for treating a thyroid gland of a total effective diameter of
4 cm using a small ionizing radiation source at its center to treat a central inner tumor
with a diameter of 1 cm, estimate with reasoning and stating assumptions what pro
portion of healthy tissue will remain if 99% of the tumor tissue is destroyed by the
treatment.
7.13 In developing a simple microfluidics-based pH sensor for biological samples, two par
allel input channels, one containing a biological fluid sample of unknown pH, and
the other containing a fluid pH indicator which changes color on mixing with the
sample, were pumped under laminar flow into a single common detection chamber.
In the first design, a color change in the detection chamber was observed only beyond
a distance of ~3 mm from the point of entry of both input flow channels, whereas in
a second design which had chevron shapes etched into the detection chamber sur
face the color changed after more like ~1 mm distance from the point of entry. Why
is this?