Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 164

technique that fi rst separates the proteins based on their sizes
[when used in the presence of sodium dodecyl sulfate (SDS)] or
their three-dimensional shapes and overall charges (when used in
the absence of SDS or reducing agents), followed by transferring
the resolved protein profi les onto a membrane (PVDF or nitrocel-
lulose), which is then used to detect specifi c protein when used in
conjunction with an antibody that recognizes the protein.
A variation of the Western blot analysis is the electrophoretic
mobility shift assay (EMSA), which was developed to study the
interaction between a defi ned fragment of DNA or RNA and
specifi c protein(s). Most currently used procedure of EMSA is
based on methods described by Garner and Revzin, 1981 and
Fried and Crothers ( 2, 3 ). Procedurally, EMSA is very similar to
Western blot analysis except that transferring the resolved
protein gel to a membrane is not absolutely necessary depending
on what the probe (DNA or RNA fragment) is conjugate with.
More details are presented in Sect. 2.2 .
When used in conjunction with Western blot analysis, immu-
noprecipitation (IP) is a powerful technique that can be used to
identify the makeup of a multiprotein complex. However, IP
requires the availability of antibody that can recognize the specifi c
protein in its native structural confi guration. The use of this anti-
body can precipitate its associating proteins to be further identifi ed
and characterized.
Finally, we also include the method chromosome immunopre-
cipitation (chIP) which is a variation of the IP technique. This
technique was developed originally by Gilmour and Lis, 1984,
1985, 1986 which was later improved and modifi ed by Solomon
et al., 1988 and Hecht et al., 1996 ( 4-9 ). This method enables the
identifi cation of fragments of DNA or RNA that can associate with
a known protein. Initially, this method is used only in the identifi -
cation of regions of chromosome that is involved in the regulation
of protein expression. However, with the current interest in epige-
netics, chIP can also be used to identify DNA modifi cation corre-
sponding to changes in total protein profi le.
2. Sample
Preparation
Much of the success of the assay is dependent on the sample prepa-
ration. In neurobiological experiments, our samples may be derived
from cell lines or tissues such as part of the brain or the spinal
cord. For cell lines, the lysis can be achieved by direct contact
of the intact cells with a detergent-based Lysis Buffer ( 10, 11 ).
The use of low detergent concentrations in a buffer to cause cell
lysis is gentle enough to enable the proteins of interest to retain
their native structure which is vital to EMSA, IP, and chIP assays.