The protocol for the isolation of nuclear and cytoplasmic fractions can be used to generate material for the gel shift/super shift/EMSA assays or any other assay requiring similar source material. Remember to use only specially prepared reagents that are free of any contaminating proteases or nucleases and to keep the preparation on ice where indicated. A common troubleshooting finding is that breakdown products caused by non-specific proteolysis are recognized in systems typically as lower molecular weight bands.
Product | Preparation |
Wash the cells gently with PBS buffer. Collect the cells by centrifugation using a microcentrifuge at 1000 rpms.
Resuspend the pellet in 5 pellet volumes of CE buffer (approximately 100 µL). Incubate on ice for 3 minutes to lyse the cells. Spin the preparation using a microcentrifuge at 1000–1500 rpms for 4 minutes. Move the supernatant containing the cytoplasmic extract to a clean tube (CE tube).Wash the nuclei (NE tube) with 100 µL of CE buffer without detergent.
Note: Be careful to resuspend the fragile nuclei gently.
Remove the supernatant and add 1 pellet volume NE buffer to the nuclear pellet (approximately 50 µL).
Adjust the salt concentration to 400 mM using 5 M NaCl (add ~35 µL). Add an additional pellet volume of NE buffer. Vortex to resuspend the pellet.Incubate the extract on ice for 10 minutes. Vortex the mixture periodically to re-suspend the pellet.
Spin the CE and NE tubes at maximum speed for 10 minutes to pellet any nuclei.Transfer the supernatant of the CE tube and NE tube separately to clean tubes. Add glycerol to the CE tube to 20%. Store at -70°C.