For subcellular fractionation:

 

Obtain fresh tissue. Cut into small pieces. Homogenize with Buffer A (see below). Dounce 10-15 strokes slowly to avoid bubbles. Let it stand on ice for 10 minutes. The following steps should be done on ice or in cold room.

 

1. Transfer homogenate into a tube labeled "Nucleus" and aliquot out 50ul into another tube labeled   "Total" . Set this tube aside on ice.

2. Centrifuge "Nucleus" lysate at 1,000g (3.5k rpm) for 10 minutes in cold room. After centrifuge you will    have a supernatant and pellet.

3. Transfer supernatant to a tube labeled "Mitochondria". Don't discard pellet, this is crude nuclear pellet.

4. Wash pellet 2x with Buffer A each time re-centrifugeing at same speed, 1,000g, for 10 minutes. Discard washes.

5. Centrifuge tube labeled "Mitochondria" at 10,000g (10.5k rpm) for 10 minutes in cold room. After centrifuge you will have a supernatant and pellet.

6. Transfer supernanant into a tube labeled "Pre-Cytoplasm". Do not discard pellet. This is mitochondrial fraction (heavy membrane fraction).

7. Wash pellet 2x with Buffer A, each time re-centrifugeing at same speed, 10,000g, for 10 minutes. Discard washes.

 

If Endoplasmic Reticulum (ER) is needed, omit step 8 and go to step 9.

 

8. Centrifuge tube labeled "Pre-Cytoplasma" at 20,000g (14k rpm) for 10 minutes to obtain cytoplasm and other light membrane fractions. Discard pellet and transfer supernatant to a new tube labeled "Cytoplasm".

9. Transfer supernatant to a tube labeled "ER" and spin 1 hour at cold room at 37k rpm (100,000g) in Sorvall fixed angle rotor (TFT80.4). Transfer supernatant to a new tube labeled "Cytoplasm". Wash pellet 2x with Buffer A at same speed for 30 minutes each.

10. Go ahead to use the samples for other experiments, or keep at -70 centigrade.

 

Buffer A: 0.25M Sucrose, 50mM HEPES, 10mM NaCl, 10nM EDTA, 2mM DTT, Protease inhibitors, 1mM PMSF)

RIPA: 1% Igepal CA-630, 0.5% sodium deoxycholate, 1% SDS in PBS, Protease inhibitor)

 

 

Refer to: Cindy Yamamoto's Protocol and Methods in enzymology, Volume 182: Guide to Protein Purification; Murray P. Deutscher (Editor),et al