We are using samples of the prefrontal cortex and hippocampus of mouse brain tissue. For protein extraction, we want to obtain a nuclear fraction, cytosolic fraction, and a synaptosomal fraction. So far, we have had a protocol to extract a cytosolic fraction and a synaptosomal fraction, but we have never needed the nuclear fraction. Now, we want to test for PPAR-alpha in a western blot, and therefore, we need the nuclear fraction. Initially, we used an isotonic buffer to maintain the integrity of the synaptosomes down the line. This was the protocol:

Homogenization buffer:

- 320 mM Sucrose

- 4 mM HEPES pH 7.4

- 2 mM EDTA

Lysis buffer:

- 50 mM Tris-HCl pH 6.8

- 1.3% SDS

- 6.5% Glycerol

1. Add protease (1:1000 or 1 tablet per max 10 ml) and phosphatase (if needed; 1:100) inhibitors cocktails to the appropriate aliquots of homogenization and lysis buffer. Place the buffers on ice. Calculate: 1:10 volumes tissue:homogenization buffer and about 200µl per sample for lysis buffer.

2. Get the tissue samples out from the -80°C freezer and place them on ice.

3. Add 1/10-1/20 (50-100µl) homogenization buffer and make sure that the tissues are completely submerged in the liquid.

4. Homogenize with a metal/plastic pestle until no debris is left. Try to avoid the formation of bubbles/foam.

5. Add the rest of the homogenization buffer and further homogenize with intense pipetting. Try to avoid the formation of bubbles/foam.

6. Centrifuge at 800g (max up to 1000g) for 15 min at 4°C (precool the centrifuge).

7. Total lysate: Transfer 10% of the supernatant into a separate tube, mix 1:1 with lysis buffer, and boil at 95°C for 5 minutes. Rest on ice for protein determination.

8. Pellet: nuclei and not well-homogenized residual tissue. Discard the pellet, or in case of low yield, save it at -80°C for further preparations.

9. Transfer the rest of the supernatant into a separate tube and centrifuge at 10000g for 15 min at 4°C.

10. Supernatant – cytosolic fraction (only if needed): mix 1:1 with lysis buffer, boil at 95°C for 5 min. Rest on ice for protein determination.

11. Synaptosomes: resuspend the pellet in 10x volume lysis buffer (about 150-250µl) by vortexing and intense pipetting. Try to avoid the formation of bubbles/foam.

12. Boil at 95°C for 5 min. Rest on ice for protein determination.

13. After protein determination, add 1/100µl DTT-blue/red solution (add up to 3x in case of high MW proteins), boil again at 95°C for 5 min, and store the samples at least at -20°C.

• After obtaining the nuclear pellet, following the first centrifugation, how should we proceed to extract the nuclear fraction? We thought we could use the following lysis buffer for extracting the nuclear fraction and mix it 1:1 with 2x Laemmli buffer after protein estimation:

- 50 mM Tris-HCl pH 7.4

- 150 mM NaCl

- 1% NP-40

- 40 mM NaF

- 10 mM EDTA

- 0.1% SDS

- 0.1% Sodium deoxycholate

- 15 mM MgCl2

- Tablet/5-10 ml protease inhibitor cocktail

• Does the nuclear pellet need to be further centrifuged to achieve good purification of the nuclear fraction?
• Do we need to use a hypotonic buffer before adding the lysis buffer?

Thank you in advance