Biomedical Engineering Reference
In-Depth Information
0.12
increased surface
expression
basal surface
expression
mock
r
2
=0.995
0.10
r
2
=0.999
0.08
0.06
0.04
0
10
20
30
Time (min)
Fig. 2
Change in absorbance at 486 nm over time in individual wells. Linear
regression was performed on the absorbance measurements of wells containing
mock transfected cells,
ʲ
-lactamase fusion construct transfected cells treated
with PBS, and
-lactamase fusion construct transfected cells treated with a drug
that increases surface expression. Note that absorbance in the mock control
wells does not change, while the absorbance in wells containing
ʲ
ʲ
-lactamase
expressing cells increases in a linear fashion
stable cell lines. This is why we detailed the generation of stable
cell lines in this chapter. Some optimization of the experimen-
tal steps might be required if one chooses to use a transient
transfection approach.
2. The assay can be miniaturized to 96-well format or smaller by
scaling down reagent volumes accordingly. However, to get
robust data it is important to use a plate-reader that can
shake
the plate between reads. Without shaking, local consumption
of substrate can affect the reaction rate.
3. The change in absorbance should be linear over time for data
analysis (Fig.
2
) Deviations from linearity could indicate: (1) that
surface expression is too high, causing saturation (
see
Note 4
),
(2) that local accumulation of product/depletion of substrate
at the bottom of the well is occurring, or (3) that surface
expression is not stable at room temperature. In the case of
local substrate depletion, which would be visible as an uneven
color change in a well, a plate reader that can shake between
absorbance reads should be used in order for the well contents
to remain homogenous. If surface expression is not stable at RT,
