Hello,
We've been trying to implement an in-house protocol for the extraction of nuclear and cytoplasmic protein fractions. We confirmed the purity of the nuclear fraction by Western blot (positive for NUP, negative for calnexin). Unfortunately, our cytoplasmic fraction consistently shows the presence of both calnexin and NUP, indicating contamination with nuclear components.
Based on this, I suspect the issue lies in the initial steps of the cytoplasmic isolation.
The cytoplasmic extration step starts after washing cells with PBS and proteinase inhibitor. Than, 150 uL of harvest buffer is added to the pellet, composed of 10 mM HEPES (pH 7.9), 50 mM NaCl, 0.5 M sucrose, 0.1 mM EDTA, and 0.5% Triton X-100. Cells are vortexed in this buffer for 2 minutes, followed by centrifugation at 1000 RPM for 10 minutes at 4°C. The supernatant should be the cytoplasmic protein fraction.
Do you have any suggestions on how we could improve this step to avoid nuclear contamination in the cytoplasmic fraction? We would greatly appreciate any advice or insights you might have, especially if you’ve encountered similar issues in your own work.
Have you ever thought of subcellular fractionation using density centrifugation, which may enhance the isolation efficacy/recovery w/o impurities?
In addition, dilute the lysate (use a bit more lysis buffer) and use RNAse/DNAse additives to reduce the overall viscosity...Increase the RPM and time of centrifugation for better cytosol isolation. These tips may also improve the nuclei pelleting performance.
Alternatively, a milder lysis buffer with physical disruption or with lysozyme addition can enhance the purity of the cytoplasmic protein supernatant.
The type and conc. of the detergent and speed of vortexing can impact the lysis of membranes thus quality of your cytoplasmic fraction. I have recently used the following protocol for HEK293T Rex cells and it worked perfectly for me. You can adopt if you like.
1. Harvest cells from 15 mm dish and wash with PBS.
2. Resuspend cells in 500 µL of hypotonic Buffer A ( 10 mM HEPES (pH 7.4), 10 mM NaCl, 1.5 mM MgCl2 and protease inhibitor cocktail) and keep in ice for 15 minutes. Some cells can be kept as whole cell lysate control and lysed by sanitation separately.
3. Add 5 µL of 10% NP40 (Final conc. 0.1%) and vortex (don't pipette) for 10 sec at low speed (I kept the speed at 4 in my vortex) followed by inverting the tubes 10 times.
4. Pellet cells at 800g for 10 min at 4 degrees and take supernatant as your cytoplasmic fraction.
5. Wash the nuclear pellet with Buffer twice and then resuspend in 250 µL of Buffer B ( 20 mM HEPES (pH 7.4), 400 mM NaCl, 1.5 mM MgCl2, 25% glycerol and protease inhibitor cocktail) and lyse by sonication.
Hope this helps and best of luck.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- DNA