Analysis of DNA-Binding Activity in Neuronal Tissue with the Electrophoretic Mobility-Shift Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 313

2. Incubate at 37°C for 30 min to 1 h.

3. Separate unincorporated nucleotides by size-exclusion chromatography (e.g.,
Nuctrap Push Columns, Stratagene).

4. Nuctrap push columns: Equilibrate the column with 80 µ L of 1X STE buffer.

Bring sample volume to 80 µ L with STE buffer, and then load onto the resin.

Elute sample with 80 µ L of STE buffer, and bring eluate to a fi nal concentration of

100 µ L, which equals 1 ng of labeled oligonucleotide/ µ L. Measure radioactivity

of 1 µ L of eluate in a scintillation counter.

5. Freeze labeled, double-stranded oligonucleotides at -20°C in a Plexiglas
container.

3.3. Preparation of Protein Extracts
3.3.1. Quick and Easy Whole Cell Protocol

This is a rapid protocol, which reduces the likelihood of post-harvest protein
modifi cations that may affect the interaction between binding proteins and
DNA. It is particularly recommended with samples in which the state of
phosphorylation of proteins infl uences the binding properties. Phosphoryla-
tion of DNA-binding proteins is fairly common and should be taken into
consideration when designing EMSA experiments. However, whole cell
extracts are not appropriate for all DNA-binding proteins. Some DNA-binding
proteins are regulated by compartmentalization, for example, they are kept
outside the nucleus and translocated to the nucleus to activate transcription
whenever necessary. In these cases it is recommended to use nuclear extracts
for EMSA ( see Subheading 3.3.2. and Note 6 ).

3.3.1.1. N EURONAL C ULTURE

1. Scrape cells in 100 µ L of sonication buffer/1 × 10 6 neurons (1 well of a 12-well

plate).

2. Sonicate for 10 s with an ultrasonic dismembrator at medium setting. Some
proteins may be sensitive to sonication. In these cases, homogenize with a
tight-fi tting glass or Tefl on-pestle homogenizer.

3. Centrifuge at 16,000 g for 10 min at 4°C.

4. Take supernatant for EMSA. Freeze aliquots of supernatant in liquid nitrogen and
store at -80°C. All samples should have the same protein concentration if you
start out with the same number of cells. Measure protein concentration in some
selected experiments to confi rm that protein measurements are not needed.

3.3.1.2. B RAIN T ISSUE

1. Dissect tissue, quick freeze, and weigh frozen tissue.

2. Add 1 µ L of sonication buffer to each 20 µ g of frozen tissue.