ReadiPrep™ Mitochondrial/Cytoplasmic Fractionation Kit

Protocol summary

1. Wash cells with PBS
2. Add 2 mL of Isolation Buffer
3. Incubate on ice for 10 minutes
4. Homogenize the cells using Homogenizer or add Reagent A.
5. Centrifuge at 7000 g for 10 mins
6. Remove the supernatent (Cytoplasmic Fraction)
7. Resuspend the pellets in Isolation Buffer
8. Centrifuge at 7000 g for 10 minutes
9. Resuspend the pellets in Assay Buffer (Mitochondria Fraction)

Important notes
Thaw all the kit components to room temperature before starting the experiment. Keep the buffer on ice during the experiment.

1. Wash the cells once with cold PBS. For cells in suspension, collect the cells by centrifugation.
   
   
2. Add 2 mL of Isolation Buffer (Component A) to cells and incubate on ice for 10 minutes.
   
   
3. Two ways cytoplasmic fraction can be extraced.
   
   Option A: Add 40 µL of Reagent A (Component B) into the mix, vortex and incubate on ice for 10 minutes. 
   
   Option B: Homogenize the cells using a Dounce Tissue Grinder by stroking 4-5 times.
   
   
4. Centrifuge the sample at 600 g for 10 minutes at 4 °C and collect the supernatent and centrifuge again at 7,000 g for 10 minutes at 4 °C. The supernatent is cytoplasmic fraction.
   
   
5. Re-suspend the pellet in 1 mL of Isolation Buffer (Component A) and centrifuge at 7,000 g for 10 minutes at 4 °C.
   
   
6. Discard the supernatent, and resuspend the pellet in the appropriate assay buffer based on downstream applications.
   
   
7. The protein concentration can be quantified by Amplite™ Fluorimetric Fluorescamine Protein Quantitation Kit (Cat# 11100).