ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit

Protocol summary

1. Rinse cells with PBS
2. Add 500 µL of Cytosol Extraction Buffer
3. Centrifuge for 20 seconds
4. Collect the supernatant (Cytoplasmic extract)
5. Re-suspend the pellets in 150 µL 1X High Salt Buffer
6. Centrifuge for 20 minutes
7. Collect the supernatant (Nuclear Extract)

Important notes
Thaw all the kit components at room temperature before starting the experiment (you may store buffers at 4°C or –20°C), keep the buffers on ice during the experiment.

1. Cytosol Extraction Buffer (1X):
Add 100 µL of 10X Protease Inhibitors (Component C) and 1 µL of DTT (Component D) into 0.9 mL of Cytosol Extraction Buffer (Component A).

2. Nuclear Extraction Buffer (1X):
Add 100 µL of 10X Protease Inhibitors (Component C) and 1 µL of DTT (Component D) into 0.9 mL of Nuclear Extraction Buffer (Component B).

Note: 0.5 mL 1X Cytosol Extraction Buffer and 150 µL of 1X Nuclear Extraction Buffer is enough for 1 assay, prepare fresh buffer as needed.

For guidelines on cell sample preparation, please visit
https://www.aatbio.com/resources/guides/cell-sample-preparation.html

1. Rinse the cells 1 time with cold PBS. For cells in suspension, collect the cells by centrifugation.
   
   
2. Add 500 µL of 1X Cytosol Extraction Buffer to cells. For adherent cells, scrape the adherent cells into a 1.5 mL centrifuge tube.
   
   
3. Vortex vigorously to fully re-suspend the cells.
   
   
4. Centrifuge at 16,000 g for 1 - 2 minutes and transfer the supernatant (Cytoplasmic extract) to another clean tube. Keep the tube on ice for downstream applications or store at -80°C.
   
   
5. Re-suspend the pellet in 150 µL 1X Nuclear Extraction Buffer.
   
   
6. Vortex vigorously to fully re-suspend the pellet.
   
   
7. Rotate the tube at 4°C for 30 minutes.
   
   
8. Centrifuge at 16,000g for 20 minutes and transfer the supernatant (Nuclear extract) to another clean tube. Keep the tube on ice for downstream applications or store at -80°C.