ReadiPrep™ Nuclear/Cytoplasmic Fractionation Kit
Example protocol
AT A GLANCE
Protocol summary
- 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)
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.
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
https://www.aatbio.com/resources/guides/cell-sample-preparation.html
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.
Citations
Authors: Ban, Dexiang and Xiang, Zhenyang and Yu, Peng and Liu, Yang
Journal: Applied Biochemistry and Biotechnology (2022): 5151--5166
Authors: Ma, Weiguo and Zhao, Xin and Gao, Yun and Yao, Xiaobin and Zhang, Junhua and Xu, Qingxia
Journal: Bioengineered (2022): 4411--4427
Authors: Li, Xinyu and Zhao, Xin and Li, Jin and Zhang, Xiaozhan
Journal: (2022)
Authors: Liu, Feng and Zhang, Xiangyang and Wu, Fei and Peng, Hao
Journal: Translational Oncology (2021): 101219
References
Authors: Gardestrom, P.; Wigge, B., Influence of Photorespiration on ATP/ADP Ratios in the Chloroplasts, Mitochondria, and Cytosol, undefined
Journal: Plant Physiol (1988): 69-76
Authors: Lilley, R. M.; Stitt, M.; Mader, G.; Heldt, H. W., Rapid fractionation of wheat leaf protoplasts using membrane filtration : the determination of metabolite levels in the chloroplasts, cytosol
Journal: Plant Physiol (1982): 965-70