Protein Analysis - Animal Models of Acute Neurological Injuries: Injury and Mechanistic Assessments - page 173

control [mixing the probe (yielded from the original DNA or
RNA fragments) with a known associating protein] and
negative control (probe alone).
5. These samples are then resolved by native PAGE before
transferring onto a membrane as described in Sect. 3.2 .
6. Since the probe is radio-labeled, the membrane is exposed
directly onto X-ray fi lm. The probe will show up as bands on
the developed fi lm indicating its mobility in each sample. The
duration of the exposure will depend on the intensity of the
radioactivity which can be estimated by the Geiger counter.

Since DNA and protein binding affi nity is highly dependent on the
environment provided by the Binding Buffer, determining and opti-
mizing the binding buffer composition is essential. However, the
optimal Binding Buffer differs with each pair of protein and nucleic
acid fragments, one should always adapt from reported Binding
Buffers used with the same category of proteins. Also, always include
a positive control (known protein that can bind to this nucleic acid
fragment) which will facilitate the optimization of the Binding
Buffer. In cases where the known proteins associating with the DNA
and RNA fragments is unavailable, it is possible to use purifi ed
nuclear protein fractions to elicit mobility shift in order to identify
key bases on the DNA or RNA fragments that are essential to pro-
tein binding. Also, to confi rm the identity of the protein that binds
to the nucleic acid fragments, specifi c antibody to the protein is
included in the mixture to produce a more signifi cant shift in
mobility for the probe which is referred to as super-shift assay.

4.3. Variations

5. Immuno-
precipitation

Immunoprecipitation is a common technique employed to identify
other proteins that are associated with the protein of interest.
However, the caveat of this technique is the need of an antibody
that can bind to the native structure of the protein of interest. The
success of this method relies essentially on preserving any protein-
protein interaction present in the cells during and after the protein
lysate is being prepared as well as the specifi city of the antibody
used. Typically, the results of IP are analyzed by Western blot anal-
ysis. In the following section, we provide details only on the
Immunoprecipitation step.

5.1. Material
and Instruments

1. Specifi c antibody to the target protein of interest.
2. TBST(TBS with 0.5% Tween 20).
3. Protein A cross-linked to sepharose or agarose beads.*
4. Sample Buffer (15 mM Tris-Cl, 6% glycerol, 0.5% SDS,
3.6 mM 2-mercaptoethanol, 0.1% bromophenol blue).

5.1.1. Materials