Fractionation
1. Scrape cells and collect by centrifugation (500 × g, 5 min, 4 °C)
2. Resuspend cell pellets in lysis buffer and incubate on ice for 10 minutes
3. Centrifuge at 500 x g for 5 min at 4C
4. Resuspend pellet in lysis buffer one drop at a time
5. Incubate on ice for 10 minutes
6. Add 2.5 volumes of sucrose buffer by pipetting slowly into wall of tube
7. Centrifuge mixture at 3500 x g, 10 min, 4C
8. Collect supernatant as cytoplasmic fraction and proceed to protein extraction
9. Keep nuclei pellets and proceed to protein extraction
Protein Extraction
1. Wash cell fractions using ice cold PBS
2. Centrifuge at 500 x g for 5 minutes and aspirate PBS
3. Add 1 mL 1XRIPA buffer (with protease inhibitor) for 1x10^7 cells
4. Agitate the contents in microcentrifuge tubes for 30 minutes at 4C
5. Centrifuge tubes at 12,000-16,000 x g for 20 minutes at 4C. Collect supernatant in fresh tubes and place on ice. Discard pellets.
6. Perform BCA assay to determine protein concentration
Mohammad Sadegh Taghizadeh what changes do you suggest that I make?
Is it a protocol from kit's instruction?
If yes, maybe need change incubation times or centrifugation xg. As I know, centrifuging a lysate at 500 xg for 10 min separate nuclei components from others. Followed by centrifugation of supernatant (from previous step) at 10000 xg for 20 min separate mitocondrial components, and centrifugation of supernatant (from previous step) at 100000 xg for 1 h separate microsomes so that supernatant is contained cytoplasmic proteins.
The use of gradient sucrose buffer separate proteins based on their sedimentation coefficient. Therefore you can choose the relevant fraction.
Generally, you must do this protocol and evaluate the results. If need you can change factors to optimization
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Molecular Methods
- Cloning