Bio-Rad Apoptosis Assays - Manual (Page 18)

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6. Aliquot samples at appropriate volumes into labeled tubes and store at
–80°C. Prepare an aliquot for protein determination. Cytosolic fractions
may be stored on ice while the nuclear + mitochondrial fractions are
prepared.
Nuclear + Mitochondrial Fraction
7. Wash the pellet from the cytosolic preparation by resuspending in
750 µl of CEB and centrifuge at 4°C for 10 min at 10,000 x g.
8. Aspirate and discard the supernatant from the tube, being careful not
to disturb the pellet.
9. Resuspend the pellet in LDB. The volume of LDB needed will be
the same as the total volume of CEB used in Table 5. Be sure to
completely disrupt the pellet by pipet mixing. Vortex for approximately
10 sec and incubate sample on ice for 15 min, vortexing for 10 sec at
each 5 min interval.
10. Centrifuge the samples at 4°C for 10 min at 10,000 x g.
11. Aspirate the supernatant and transfer to a 1.5 ml snap cap tube
labeled “N + M Fraction”. Be sure not to disturb the pellet when
aspirating the supernatant. Discard the pellet. Aliquot samples at
appropriate volumes into labeled tubes, store at –80°C, and prepare
an aliquot for protein determination.
12. Determine the protein concentration of the samples using the Bio-Rad
protein assay reagent (Bio-Rad cat #500-0006) or equivalent and
diluting samples 1:20 in PBS.
13. For sample analysis, bring samples to final concentrations of
250 µg/ml for Apoptosis Panel 1 and 500 µg/ml for Apoptosis Panels
2 and 3 by diluting with LDB. Additional dilutions may be required
depending on sample.
Preparation of Total Extracts
1. Prepare the LDB by adding 1 tablet each of the protease inhibitors
listed above to 10 ml of buffer. Once the tablets are in solution, store
the buffer at 4°C for up to 1 week or at –20°C for up to 3 months.