Surfactant-Free Nuclear Protein Extraction Protocol
Product Manager：Harrison Michael
In the process of nuclear protein extraction, the use of surfactants can significantly interfere with protein labeling efficiency. This is because surfactants can alter the conformation of proteins and may interact with the target protein, affecting its biological activity and the accuracy of subsequent analyses. Therefore, adopting a surfactant-free extraction protocol ensures the acquisition of purer, more stable nuclear protein samples, which in turn enhances the reliability of experimental results. Moreover, surfactant-free extraction methods typically preserve the native state of proteins better, allowing them to function more closely to physiological conditions in subsequent experiments. This strategy is applicable not only to the extraction of nuclear proteins from cultured cells but also to the processing of tissue samples, further improving the protection of protein activity during extraction.
From Adherent Cells to Extract Nuclear Proteins
1. Cell Culture:
- Culture cells to 70-80% confluence, ensuring appropriate media and developmental conditions (such as temperature and CO₂ concentration) to support cell development.
2. Remove Culture Media:
- Carefully remove the cell culture media to avoid damaging the cells, ensuring that the cells remain moist after media removal to prevent drying.
3. PBS Wash:
- Wash the cells twice with phosphate-buffered saline (PBS), taking care not to detach the cells. After washing, gently shake the conical centrifuge tube to ensure the cells are completely washed. Discard the PBS, scrape the cells with fresh PBS, and transfer them to a conical centrifuge tube.
4. Centrifuge:
- Centrifuge at 450 × g for 5 minutes, discarding the supernatant.
5. Estimate Cell Volume:
- Estimate the packed cell volume (PCV) and record the data.
6. Prepare Lysis Buffer:
Prepare 1 mL of lysis buffer (5 × PCV), including:
- 10 mM HEPES (pH 7.9)
- 1.5 mM MgCl₂
- 10 mM KCl
- Add 1% (v/v) protease inhibitor (such as PMSF or others). Ensure all reagents are of high purity to avoid contamination.
7. Resuspend Cells:
- Resuspend the cell pellet in the lysis buffer, mixing gently to avoid foam formation. Use gentle repeated pipetting during resuspension to ensure sample uniformity.
8. Incubate:
- Incubate in the lysis buffer for 15 minutes to promote cell swelling.
9. Centrifuge Pellet:
- Centrifuge at 420 × g for 5 minutes, discarding the supernatant.
10. Resuspend Pellet:
- Resuspend the cell pellet in 400 µL (2 × PCV) of lysis buffer.
11. Cell Disruption:
- Use a glass tissue homogenizer or a syringe with a 27-gauge needle to disrupt the cells. Slowly aspirate and expel the suspension, repeating five times to ensure complete cell lysis.
12. Check Lysis Effectiveness:
- Observe the lysis status. Add trypan blue to the cell sample to confirm the extent of cell lysis. Intact cells do not absorb the dye, while lysed cells will stain the nuclei. Consider using a microscope to assess the degree of cell lysis.
13. Centrifuge to Separate:
- Centrifuge at 10,000-11,000 × g for 20 minutes to separate cell debris.
14. Extract Cytoplasm:
- Transfer the supernatant to a new centrifuge tube to obtain the cytoplasmic fraction.
15. Extract Nuclear Proteins:
Prepare the extraction buffer:
Add 1.5 µL of 0.1 M DTT and 1.5 µL of the protease inhibitor mixture to 147 µL of extraction buffer.
Resuspend the crude nuclear pellet in 140 µL of extraction buffer, gently shaking for 30 minutes to promote nuclear extraction.
16. Final Centrifugation:
- Centrifuge at 20,000-21,000 × g for 5 minutes, transferring the supernatant to a cooled tube for dialysis or storage at -70°C.
From Tissue to Extract Nuclear Proteins
1. Tissue Handling:
- Wash 100 mg of tissue twice with PBS, discarding the PBS. Ensure tissue samples are kept on ice before processing to reduce degradation.
2. Lysis Buffer:
Prepare a low-osmolarity lysis buffer:
Add 14 µL of 0.1 M DTT and 14 µL of the protease inhibitor mixture to 1,400 µL of lysis buffer.
3. Homogenize Tissue:
- Gently resuspend the tissue in 1 mL (5 × PCV) of lysis buffer containing DTT and protease inhibitors using a tissue homogenizer. Take care to avoid overheating during homogenization, which may affect protein stability.
4. Centrifuge:
- Centrifuge at 10,000-11,000 × g for 20 minutes to obtain the cytoplasmic fraction.
5. Nuclear Extraction:
- Resuspend the crude nuclear pellet in 140 µL (2/3 × PCV) of extraction buffer containing DTT and protease inhibitors, gently shaking for 30 minutes.
6. Final Centrifugation:
- Centrifuge at 20,000-21,000 × g for 5 minutes, transferring the supernatant to a clean cooled tube for dialysis or storage at -70°C.
Dialysis Steps
1. Prepare Dialysis Buffer:
- Prepare a 0.1 M bicarbonate buffer (pH 9.5-9.6).
2. Dialyze:
- Dialyze the extract at 4°C with a dialysis buffer volume 1000 times the volume of the nuclear protein extract for 2 hours.
3. Change Buffer:
- Replace the dialysis buffer with freshly prepared bicarbonate buffer and continue dialysis at 4°C for another 2 hours.
4. Protein Concentration Determination:
- Measure the protein concentration using the Bradford method, ensuring the extract reaches the desired pH before the labeling procedure.
Conclusion
In summary, employing a surfactant-free nuclear protein extraction protocol not only avoids interference with protein labeling and activity but also maintains the structural integrity of nuclear proteins, thereby enhancing the accuracy and reliability of experiments. Through meticulous steps and the use of high-purity reagents, researchers can obtain high-quality nuclear protein extracts that lay a solid foundation for subsequent functional analyses and biological studies.
Aladdin：https://www.aladdinsci.com/