Subcellular Fractionation from Fresh Tissue
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
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