Biology Reference
In-Depth Information
where nonreducing native PAGE is preferred, the 2-mercaptoethanol
is also removed from the Sample Buffer. Under such circumstances,
proteins are resolved based on their shape and charges. The advantage
of native PAGE is in the study of protein complexes where each
complex resolves as individual entities. Consequently, we can deter-
mine whether the protein of interest exists on its own or as part of
a complex under physiological conditions.
4. Electrophoretic
Mobility Shift
Assay
EMSA is an assay developed to examine the association between
DNA or RNA fragments and specifi c proteins. In many studies,
this method is used mainly to study key bases on the DNA or RNA
fragments that alter their binding affi nity to protein(s).
4.1. Material and
Instruments
1. Binding Buffer (5 mM HEPES pH 8.0, 85 mM KCl, 0.5%
NP-40, and protease inhibitor cocktail)
2. Random prime labeling kit (Invitrogen) for labeling the DNA
or RNA fragments
3. Purifi ed protein known to associate with this DNA or RNA
fragment
4. Purifi ed protein(s) of interest
5. [a-P 32 ]dCTP
4.1.1. Materials
1. Water bath
2. Geiger counter
4.1.2. Instruments
4.2. Procedures
1. First, we need to generate radio-labeled probes, which are the
radio-labeled version of the DNA or RNA fragments to be
examined, using the random prime labeling system (Invitrogen).
Please refer to manufacturer's manual for more detail
instructions.
2. In brief, the random prime reaction is a polymerase reaction
using each of the DNA or RNA fragments as template in turn,
in the presence of radio-labeled [a-P 32 ]dCTP and mixture of
dATP, dTTP, and dGTP with random hexameric nucleotides
as primers.
3. After the polymerase reaction, the resultant probe is separated
from the residual [a-P 32 ]dCTP via column purifi cation giving
rise to the probes required in EMSA.
4. Once the probes are ready, they are mixed individually with
the protein(s) of interest in Binding Buffer followed by 1-h
incubation at 37°C to allow association between the nucleic
acid fragments and proteins. It is important that a positive
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