Biology Reference
In-Depth Information
2.1.5.2.3
Preparation of luciferase assay
50
m
L per sample
3 mL of luciferase assay reagent are prepared just before use. For
1 mL, the luciferase assay reagent contains 500
m
L2
þ
luciferase buffer (40 mM
Tris-phosphate, 2.14 mM MgCl
2
, 5.4 MgSO
4
, 0.2 mM EDTA, 66.6 mM DTT,
pH 7.8; stable at
20
C), 47 mL luciferin 10
2
M, 53 mL ATP 10
2
M, 27 mL
coenzyme A (lithium salt) 10
2
M, and 430 mL distilled water. This buffer is
light-sensitive.
2.1.5.2.4
Luciferase measurement
10
m
L of lysate is distributed per well of a 96-well white plate. Wash the injector and
prime with the luciferase assay reagent. The luminometer is programmed to perform
2 s premeasurement delay followed by a 10 s measurement period with 50
m
L lucif-
erase assay reagent. The measurement is performed and the luciferase activity mea-
surement is recorded.
2.1.5.2.5
Measurement of bgal activity
Per sample, the preparation of
b
gal assay buffer requires the mix of 150
m
L
b
gal
buffer (Na
2
HPO
4
(12H
2
O) 60 mM, NaH
2
PO
4
40 mM, KCl 10 mM, MgCl
2
(6H
2
O) 1 mM, and
b
-mercaptoethanol 50 mM) and 30
m
L ONPG (4 mg/mL).
10 mL lysate is distributed in a 96-well plate and 180
m
Lof
b
gal assay buffer is
added. The plate is incubated at 37
C and the time until the yellow color has devel-
oped is measured. The reaction is stopped by adding 75
m
Lof1MNa
2
CO
3
(bubbles
have to be avoided). The absorbance is immediately read at 420 nm in a plate reader
(
b
gal unit
¼
100
total volume
absorbance/(assay volume
time (in hours)).
2.1.5.2.6
Normalization
The luciferase values are normalized for transfection efficiency with the
b
gal values.
2.1.6
Fluorescence cross-correlation spectroscopy to measure the
concentrations and interactions of NRs in living cells
Novel fluorescence microscopy techniques have advanced the frontiers of quantita-
tive measurements in live cells. Among these approaches, fluorescence fluctuation
and correlation spectroscopy is especially appealing for situations in which one
wishes to measure the degree of interaction between two protein partners. This ap-
proach involves simultaneous excitation, using a variety of strategies, among them a
femtosecond-pulsed infrared (IR) laser for simultaneous two-photon excitation of
two biomolecules bearing two fluorescent dyes (
Bacia, Kim, & Schwille, 2006
).
The time correlation of their intensity in two different detection channels, along with
the autocorrelation of their individual fluorescence in single channels, allows calcu-
lation of the total concentration of each species, free and bound, and, hence, the de-
gree of complex formation between two proteins. These measurements can also
allow for the determination of complex stoichiometries. Two-photon, two-color,
fluorescence cross-correlation spectroscopy (FCCS) in transiently transfected cells
