Preparation of Nuclear Extracts for Gel Shifts and Westerns Blots
The following protocol is optimized for about 1x107 cells (near confluent 10 cm plate). NOTE: ALL COMPONENTS (BUFFERS, PROTEASE INHIBITORS, ETC) MUST BE KEPT ON ICE DURING THE ENTIRE PROCEDURE.
Buffer A:
10 mM HEPES, pH 7.9
10 mM KCl
0.1 mM EDTA
Add just before use: 1mM DTT, 0.5 mM PMSF, 5 ul of 10 ug/ul of aprotinin, leupeptin, and pepstatin A to 5 ml of buffer.
Buffer B:
20 mM HEPES, pH 7.9
0.4 M NaCl
1 mM EDTA
10% Glycerol
Add just before use: DTT, PMSF, aprotinin, leupeptin, pepstatin A as above.
1. Wash plates with PBS two times.
2. Prepare 5 ml of Buffer A in a 15 ml conical c’fuge tube, and add the following:
-5 ul of each protease inhibitor (leupeptin, PMSF, aprotinin, pepstatin, and DTT)
-200 ul of 10% IGEPAL.
Add 0.5 ml of this preparation directly to each plate, and wait 10 min. at room temp.
3. Scrape cells with NEW sterile scraper, then pipet up and down with P1000 several times to disrupt cell clumps. NOTE: you will have nearly 1 ml of lysate at this point.
4. Transfer this lysate to a 1.5 microcentrifuge tube, place on ice.
5. Centrifuge at 4 C at top speed (15000 x g) for 3 min.
6. Place tubes on ice.
7. Save supernatant (cytosolic fraction) for luciferase/ß-gal assay, if desired. Otherwise, discard the supernatant.
8. Prepare 1 ml of Buffer B in a 1.5 ml eppendorf, and add 1 ul of each protease inhibitor as above, but this time DO NOT add IGEPAL. Add 150 ml of this buffer to each tube, and resuspend pellet by pipeting up and down with a P200. Place on ice.
9. Shake vigorously at 4 C for 2h.
10. Centrifuge at 4 C, top speed for 5 min. Measure Bradford (protein) concentration using 5 ul of each sample, then aliquot 15 ul aliquots into prechilled 0.5 ml prelabeled microcentrifuge tubes, freeze in liquid nitrogen, and store in –80 C freezer.
