Transfer the tissue to a mortar at room temperature and drizzle immediately with 300 pi of grinding buffer per 1 ml of tissue while still frozen The buffer should freeze on contact with tissue

3. When the tissue has partially thawed and is slightly crunchy, but not mushy, grind thoroughly with pestle. The mixture should be stiff— neither a powder nor a slurry—while grinding.

IMPOR "ANT Move through the next four steps quickly and gendy to keep nuclear lysis toair nmum.

4. Once the tissue has thawed, use a wide-bore pipettor, or an automatic pipettor with a cut-off 1-ml pipette tip, to distribute -400 [il of tissue into 1.5-ml microcentrifuge tubes. Store on ice.

5. Centrifuge at 6000g for 3 minutes at 4°C in a microcentrifuge. Discard the supernatant and anything that floats.

6. Use a soft bristle paint brush to gently resuspend the pellet in washing buffer in a volume equal to the pellet.

7. Centrifuge at 6000g for 3 minutes at 4°C in a microcentrifuge.

8. Resuspend the pellet in an equal volume of 2x lysis buffer.

9. Mix by rocking or rotating for 30-40 minutes in a cold room. In addition, gently vortex every 10 minutes.

10. Centrifuge at 6000g for 3 minutes at 4°C in a microcentrifuge.

11. Collect the supernatant and centrifuge at 180,000g for 90 minutes at 6°C in an ultracentrifuge.

12. Carefully collect cleared supernatant and dialyze against appropriate buffer at 4°C.

The SlideALyser (Pierce) speeds up dialysis time to -1 hour if the content of the slide is mixed every 15 minutes.

13. Determine the total protein concentration in each sample, using any conventional method (e.g., the Bradford [1976] assay).

14. Snap-freeze the nuclear extracts in small quantities in liquid nitrogen. Store at -80°C.

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