In vivo ubiquitination of Post-cytosal fractionation
Materials:
TSD (50 mM Tris pH 7.5, 2% SDS, 5 mM DTT )*ADD 1:200 of 1M DTT FRESH.
II. Complete Triton X-100 Lysis Buffer as in protocol Pro 003 “Preparing Cell
Lysates”
I.
III:
Composition
HEPES
KCl
MgCl2
PMSF
DTT
Aprotinin
Leupeptin
Buffer 1 TV 25mL
[Final]
Amount
0.025M
0.1488g
5mM
125µL
0.5mM
12.5µL
1mM
250µL
1mM
250µL
1ng/mL
25µL
10ng/mL
25µL
[Stock]
Pure
1M
1M
0.1M
1M
1µg/mL
10µg/mL
Buffer 2 TV 12.5mL
Mix 12.25mL of Buffer 1 + 250µL NP-40
Buffer 3 TV 50mL
1:1 Mixture of Buffer 1 + Buffer 2
Composition
HEPES
Sucrose
NaCl
PMSF
DTT
Aprotinin
Leupeptin
Buffer 5 TV 10mL
[Final]
Amount
0.025M
59.6mg
10%
1g
350mM
700µL
1mM
100µL
1mM
100µL
1ng/mL
10µL
10ng/mL
10µL
[Stock]
Pure
Pure
5M
1M
1M
1µg/mL
10µg/mL
Procedure:
A. Fractionation:
1. Discard media and add 1 mL of ice cold PBS.
2. Scrape cells into PBS, transfer to eppendorf tube and spin at 2,500 rpm for 5 min.
3. Discard supernatant and resuspend pellet in 200µL of Buffer 1.
4. Add 200µL of Buffer 2 and rotate at 4 degrees for 15 minutes.
5. Spin at 2500 rpm for 1 minute.
6. Transfer supernatant to new microfuge tube (cytoplasmic protein).
7. Add100µL of Buffer 3 and gently mix. Spin at 2500 rpm for 1 minute.
8. Transfer the wash to cytoplasmic protein preparation from step 6. Optional
9. Suspend the pellet in 2x pellet volumes of Buffer 5 and vertex (For MEFs cells,
35ul is preferred for 15cm dish culture). Add equal volume of TSD (35ul) and
mix up by vertex. Heat at 90 ºC for 5 mins.
10. Bring up the volume of the lysate to 500 µl (250ul) by Triton X-100 Washing
buffer. And sonicate the samples in Output 2, 90% duty cycle with 20 to 30
pulses. Test the shred of the DNA by aspirating the lysate up and down with p200
pipetman. Completely sonicated samples should not give any resistance to
aspiration.
11. Add another 1mL(500ul) Triton X-100 Washing Buffer to dilute SDS down to
0.1% so the samples could be applied for immunoprecipitation.
12. After IP, rinse beads 4times/10min each at 4oC. Elute protein with LDS loading
buffer. Western then probe with ubiquitin Ab.