Sample Preparation Protocols
Cell Fractionation Procedure:
1. Decant the media from the plate.
2. Wash the cells with warm 1X PBS once.
3. Trypsinize the cells at 37oC.
4. Harvest cells (Spin at 1000Xg for 2min).
5. Wash the cells once or twice with cold PBS.
6. Measure the packed cell volume (PCV).
7. Add 10 PCV volume of ice cold hypotonic buffer (containing 1X protease inhibitor and 1X phosphotase inhibitor)
8. Incubate on ice for 10min.
9. Spin the cells at 1000Xg for 2min and discard the supernatant.
10. Add 2 volume of ice cold hypotonic buffer (containing 1X protease inhibitor and 1X phosphotase inhibitor)
11. Break the swollen cells using a dounce homogenizer.
12. Monitor the cells by trypan blue by phase microscopy to check for completeness of cell lysis. (More than 95% cells have to be broken, but the nucleus must be visible intact. If less than 95% cells broken, then repeat step 7 and keep monitoring)
13. Spin at 3000Xg for 10min and collect the supernatant. The supernatant is the Cytosolic fraction.
14. For Nuclear Extract, add 2 volumes of NETN (containing 1X protease inhibitor and 1X phosphotase inhibitor) to the pellet.
15. Sonicate the above pellet (30sec bust, 59 sec off, repeat 6 times)
16. Spin the nuclear and cytosolic fraction at 100,000Xg for 20min at 4oC.
17. Remove and store the supernatant in -80 oC.
18. For Membrane fraction, add 2 volumes of NETN containing 1X protease inhibitor and 1X phosphotase inhibitor to the 100,000X g pellet from cytosolic fraction and sonicate for 30sec bust, 59 sec off, repeat 6 times. Spin the sonicated lysate at 100,000Xg for 20 min at 4oC. Remove supernatant and save in -80 oC.
19. For Chromatin binding fraction, add 1 volumes of NETN containing 600mM KCl, 1X protease inhibitor and 1X phosphotase inhibitor to the 100,000X g pellet from nuclear extraction fraction and sonicate for 30sec bust, 59 sec off, repeat 6 times. Spin the sonicated lysate at 100,000Xg for 20 min at 4oC. Remove supernatant and save in -80 oC.
Immuno-precipitation:
1. Thaw the cytosolic/nuclear extract at 37oC.
2. Spin at 200,000Xg for 20min at 4oC and then transfer supernatant to fresh tube.
3. Add 5 µg of antibody to the above supernatant.
4. Incubate for 2 hour at 4oC, rocker shaking.
5. Spin at 100,000Xg for 15min at 4oC. Collect the supernatant
6. Add 30µl of protein A bead slurry to the above supernatant.
7. Incubate the above beads for 1hour at 4oC, inverted rotation.
8. Spin at 1000Xg for 1min and then discard the supernatant.
9. Wash the beads with 1ml NETN buffer three times, quickly. (NETN: 50mM Tris pH 7.3, 170mM NaCl, 1mM EDTA, 0.5% NP-40)
10. Spin at 1000Xg for 1min and then discard any supernatant.
11. Add 15µl of 2X SDS loading dye to the beads.
12. Heat samples for 8-10 min at 90°C.
13. Briefly spin the samples with the beads and keep it on ice.
In-gel Digestion:
1. Load samples onto NuPage 10% Bis-Tris Midi-gel (Cat. No. WG1201BX10)
2. Run up to 1/4th using MOPS buffer (Cat. No. NP0001) at constant of 80V
3. Fix gels and stain with Commassie blue.
4. Destain the gel and keep the gel in water.
5. Scan the gel and then cut gel slices depend on protein size
6. De-stain each band completely
7. Incubate gel slices in water (can keep in water O/N or change twice with 1 hr gap)
8. Dehydrate gel with 75%ACN(1hr)
9. Change pH of gel basic using 50mM ammonium bicarbonate( ABC) soln (30min*2)
10. Remove ABC soln and add 100ng/ul Trypsin(GenDepot) to each tube
11. Incubate at 37 C for over night
12. Acidify the digest by adding 20ul of 2% FA, stand for 4-5 min
13. Extract peptide from gel by adding 200ul of 100% ACN, shake (10-15min), and centrifuge at top speed (Table-top) collect the supernatant in new tubes.
14. Vacuum-dry the samples.
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