Biology Reference
In-Depth Information
The accelerating voltage (
V
c
) must be set to 50 V in order to preserve ternary
complex formation and good mass accuracy. ESI-MS data are acquired in the positive
ion mode on the mass range 1000-5000 m/z. The relative abundance of the different
species present on ESI mass spectra from their respective peak intensities is mea-
sured, assuming that the relative intensities displayed by the different species reflect
the actual distribution of these species in solution.
2.1.4
Monitoring NR-NR interactions by electrophoretic mobility
shift assays
The ability of NRs to bind to DNA as homodimers and heterodimers can be assayed
by electrophoretic mobility shift assays (EMSAs). Solutions of protein and nucleic
acid are combined and the resulting mixtures are subjected to electrophoresis under
native conditions through polyacrylamide gel. After electrophoresis, the distribution
of species containing nucleic acid is determined, usually by autoradiography of
32
P-labeled nucleic acid. In general, protein-nucleic acid complexes migrate more
slowly than the corresponding free nucleic acid. A major advantage of studying
protein-DNA interactions by an electrophoretic assay is the ability to resolve
complexes of different stoichiometry or conformation. It is also useful for studying
higher-order complexes containing several proteins, observed as a “supershift”
assay. Another advantage is that target proteins may be obtained from a crude nuclear
or whole cell extract,
in vitro
transcription product, or a purified preparation from
E. coli
or mammalian expression systems. On the other hand, identification of the protein
bound to the probe can be accomplished by adding specific antibody in the binding re-
action. If the protein of interest binds to the target DNA, the antibody will form an an-
tibody-protein-DNA complex, further reducing its mobility relative to unbound DNA.
In some cases, the antibody may disrupt the protein-DNA interaction, resulting in loss
of the characteristic shift but not causing a “supershift.” Here, a protocol is given as
example of EMSA to determine the effect of a singlemutation of tyrosine 402 to alanine
in helix H9 of RXR
a
on the RXR
a
dimerization function. This mutation of RXR
a
sub-
stantially weakens RAR
a
heterodimerization while concomitantly increasing homodi-
merization (
Vivat-Hannah, Bourguet, Gottardis, & Gronemeyer, 2003
;
Fig. 2.5
).
2.1.4.1
Required materials
- TNT rabbit reticulocyte lysate system (Promega)
-
32
P-DR5 (5
0
-TCGAGGGTAG
GGGTCA
CCGAA
AGGTCA
CTCG-3
0
; direct
repeat underlined) oligonucleotide
- Binding buffer: (10 mM Tris-HCl (pH 8.0), 0.1 mM EDTA, 0.4 mM DTT, 5%
glycerol)
2.1.4.2
Protocol
2.1.4.2.1
transcription-translation
DNA-binding proteins generated by cell-free expression systems offer an excellent
format for performing EMSA assays. Recombinant proteins (mouse RXR
a
, mutant
mouse RXR
a
Y402A, and human RAR
a
) are prepared by
in vitro
transcription-
translation using the TNT rabbit reticulocyte lysate system programmed with
In vitro
