ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit
|Shipping||Standard overnight for United States, inquire for international|
|H-phrase||H303, H313, H333|
|Intended use||Research Use Only (RUO)|
|R-phrase||R20, R21, R22|
AT A GLANCE
- Rinse cells with PBS
- Add 500 µL of Cytosol Extraction Buffer
- Centrifuge for 20 seconds
- Collect the supernatant (Cytoplasmic extract)
- Re-suspend the pellets in 150 µL 1X High Salt Buffer
- Centrifuge for 20 minutes
- Collect the supernatant (Nuclear Extract)
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.
PREPARATION OF WORKING SOLUTION
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
SAMPLE EXPERIMENTAL PROTOCOL
- Rinse the cells 1 time with cold PBS. For cells in suspension, collect the cells by centrifugation.
- Add 500 µL of 1X Cytosol Extraction Buffer to cells. For adherent cells, scrape the adherent cells into a 1.5 mL centrifuge tube.
- Vortex vigorously to fully re-suspend the cells.
- 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.
- Re-suspend the pellet in 150 µL 1X Nuclear Extraction Buffer.
- Vortex vigorously to fully re-suspend the pellet.
- Rotate the tube at 4°C for 30 minutes.
- 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.
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