Analysis of DNA-Binding Activity in Neuronal Tissue with the Electrophoretic Mobility-Shift Assay - Drugs of Abuse: Neurological Reviews and Protocols - page 314

3. Sonicate for 10 s with an ultrasonic dismembrator at medium setting, and make
sure no tissue clumps remain. Some proteins may be sensitive to sonication. In
these cases, homogenize with a tight-fi tting glass or Tefl on-pestle homogenizer.

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

5. Take supernatant, measure protein, and equalize protein concentration between
samples by adding sonication buffer with all additives to a fi nal concentration
of 2 µ g of protein/ µ L.

6. Proceed to EMSA or quickly freeze aliquots in liquid nitrogen and store at
-80°C. Repeated freeze-thaw can change the binding properties of DNA-binding
proteins. Freezing multiple aliquots of each sample is strongly recommended.

3.3.2. Crude Nuclear Extracts

This protocol is recommended if nuclear proteins need to be separated
from cytosolic proteins. It is based on refs. 1-4 . Tissue must be fresh and not
previously frozen. In the fi rst step, cells are swollen in low-salt buffer and
disrupted in a homogenizer. Nuclei should stay intact. Nuclei are then pelleted,
separated from cytoplasmic proteins, and nuclear proteins are extracted in a
high-salt buffer. Membranes are pelleted and discarded. All solutions need
to be ice cold.
3.3.2.1. N EURONAL C ULTURE (5)

of buffer A.
2. Incubate on wet ice for 10 min to swell cells.
3. Homogenize with a loose-fi tting (dounce B) homogenizer, 15 strokes up and
down.
4. Incubate on wet ice for 5 min.
5. Proceed to step 6 of the brain tissue protocol.
3.3.2.2. B RAIN T ISSUE (6)
1. Dissect brain area of interest.
2. Chop tissue into small pieces and suspend in 1 mL of buffer A; pipet up and
down with a 1-mL pipet to suspend tissue further.
3. Incubate on wet ice for 10 min.
4. Homogenize with a dounce B homogenizer, 15 strokes up and down.
5. Incubate on wet ice for 5 min.
6. Centrifuge at 4°C at 800 g for 10 min.
7. Discard supernatant (or keep if you want to analyze the cytoplasmic fraction).
8. Resuspend pellet in equal volume of sonication buffer plus additives, and
carefully resuspend with a 1-mL pipet.
9. Incubate for 30 min on wet ice.
10. Centrifuge at 16,000 g , 10 min, 4°C.
11. Take supernatant and measure protein, and discard the pellet.
12. Dilute protein with sonication buffer to 1

1. Harvest fresh cultured cells in 500 µ

µ g/1 µ L, and mix well.