Denatured Yeast Extracts
Note: Some proteins will crash out of sample buffer if boiled (particularly large, TM
proteins). Never heat these to more than 60ºC.
For all four methods, it is key to work as fast as possible.
I.
NaOH pre-incubation
This is the fastest and most reproducible method, but one you don mint
want to use if 5 min of NaOH treatment in living or azide-killed cells will
change your protein’s migration/amount on gels.
1.
2.
3.
4.
5.
Spin down 2.5 OD of cells (15 sec in microfuge)
Resuspend in 200µl 0.2M NaOH
Incubate 5 min on bench, while heating up boiling water bath.
Spin down (15 sec in microfuge).
Resuspend in 50µl 1x SDS sample buffer and place immediately in
boiling water bath.
6. Boil 3 min.
7. Spin 1 min in microfuge.
8. Load 15µl supernatant on gel.
II.
NaOH and TCA. This method works very robustly for large proteins
1.
2.
3.
4.
5.
6.
7.
Spin down 2.5 OD of cells (15 sec in microfuge)
Resuspend in 150µl 2M NaOH/150mM BME
Incubate 15 min on ice.
Add 150µl 10% TCA.
Incubate 30 min on ice.
Spin 5 min at 14,000 rpm in cold room.
Remove supernatant and resuspend by scraping on eppendorf rack in
50µl 80mM Tris pH 8.0/8mM EDTA/120mM DTT/3.5% SDS/0.15%
glycerol/0.08% w/v Tris base/ 0.01% bromophenol blue.
8. Incubate 30 min at 42ºC. Spin 1 min in microfuge. Load 5-15µl
supernatant on gel.
III.
Bead-beating
This is the classic method, but it can be difficult to get reproducible lysis,
and sensitive proteins can be degraded.
1. Spin down 2.5 OD of cells (15 sec in microfuge). Heat up boiling
water bath.
2. Resuspend in 100µl pre-heated 3x sample buffer. Place immediately in
boiling water bath.
3. Boil 3 min.
4. Add glass beads to the meniscus. Vortex 1-2 min.
5. Add 100µl 3X sample buffer. Boil 2 min.
6. Vortex for a few seconds.
7. Spin 1 min in microfuge. Transfer supernatant to a new tube. Load
15µl on gel.
IV.
TCA precipitation
This method works well for large, protease-sensitive proteins.
1.
2.
3.
4.
5.
6.
7.
Spin down 2.5 OD of cells ((15 sec in microfuge)
Resuspend in 100µl PBS. Add 25µl 100% TCA.
Freeze on dry ice or at –80ºC for 15 min to overnight.
Add 1ml 10% TCA and invert tube until frozen material thaws.
Incubate 15 min on ice.
Spin 10 min in microfuge at 4ºC.
Decant supernatant. Add 1ml ice-cold acetone. Spin 5 min in
microfuge at 4ºC.
8. Decant supernatant and remove all traces of acetone. Do not let pellet
dry out for too long!
9. Resuspend in 50µl 250mM Tris pH 8.0 by scraping on eppendorf rack.
10. Add 25µl 3x sample buffer and boil for 1 min.
11. Spin 1 min in microfuge at room temperature. Load 15µl of
supernatant on gel.