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Immunoprecipitation of Soluble Proteins from Yeast Cell Extracts
Solutions
PIPES/KOH buffer
– 0.035 g Na-azide
– 28 ml dH2O
–
7 ml 0.5 M PIPES/KOH pH 9.4
– 350 µl 1M DTT
(fresh) |
Makes 35 ml = 100 mM PIPES/KOH pH 9.4, 10 mM DTT, 0.1% Na-azide
Kpi/sorbitol buffer
– 22.4 ml dH2O
– 1.4 ml 1M K2HPO4
–
347 µl 1M KH2PO4
– 10.5 ml 2 M sorbitol
– 350 µl 1 M DTT (fresh) |
Makes 35 ml = 50 mM KPi pH 7.4, 0.6 M sorbitol, 10 mM DTT
Spheroblast wash buffer
– 25 ml dH2O
– 1.75 ml 1M HEPES/KOH pH 7.5
– 1166 µl 3 M KCl
– 87.5 µl 1 M MgCl2
– 7 ml 2 M sorbitol |
Makes 35 ml = 50 mM HEPES/KOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2, 0.4
M sorbitol
Protease inhibitors
– leupeptine (1000X):
3 mg/1.5 ml H2O
– benzamidine(1000X): 0.469 g/1.5
ml H2O
– aprotinin (1000X): 3 mg/1.5 ml H2O
–
bacitracin (1000X): 0.3 g/1.5 ml H2O
– pepstatin (1000X):
3 mg/1.5 ml MeOH
– PMSF (250X) : 0.436g/10 ml anhydrous
EtOH |
Zymolase
– 40 mg zymolase T-100
(ICN Biomedicals)
– 2ml 1M sorbitol |
EB buffer
– 45.8 ml dH2O
– 2.5 ml HEPES/KOH 1 M, pH
7.5
– 1665 µl KCl 3 M
– 125 µl
MgCl2 1M
– 50 µl DTT 1M
– 200 µl
leupep (1000X)
– 200 µl benzamid (1000X)
– 200 µl aprot (1000X)
– 200 µl
bacitrac (1000X)
– 200 µl pepst A (1000X)
– 100 µl NaF 1M
– 5 protease inhibitor
tablets (EDTA free, Roche)
– PMSF (250X), add freshly |
Makes 50 ml = 50 mM HEPES/KOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2
EBX buffer
– 47 ml EB buffer
– 1175 µl 10% Triton
X-100 |
Makes 47 ml = 50 mM HEPES/KOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2, 0.25%
Triton X-100
EBX-S buffer
– 2.9 ml dH2O
– 250 µl 10% Triton X-100
– 6 ml sucrose 50%
– 332 µl KCl 3M
– 500 µl HEPES/KOH 1 M, pH 7.5
– 25 µl
MgCl2 1 M
– 10 µl DTT 1 M
– 40 µl
leupep (1000X)
– 40 µl benzamid (1000X)
– 40 µl aprot (1000X)
– 40 µl bacitrac
(1000X)
– 40 µl pepst A (1000X)
–
20 µl NaF 1M
– 2 protease inhibitor tablets
(EDTA free, Roche)
– PMSF (250X), add freshly |
Makes = 50 mM HEPES/KOH pH 7.5, 100 mM KCl, 2.5 mM MgCl2, 0.25% Triton
X-100, 30% sucrose
Procedure
- Grow 250 ml of yeast culture until OD = 0.5.
- Harvest cells (3000 rpm, 10 min, 4° C).
- Resuspend in 5 ml PIPES/KOH buffer.
- Incubate 10 min at room temperature.
- Spin for 3 min at 2000 rpm, decant supernatant.
- Resuspend in 5 ml Kpi/sorbitol buffer.
- Take 10 µl, dilute in 990 µl dH2O in
cuvette and measure OD (600 nm).
- Add 10 µl zymolase T-100
- Incubate at 37° C for 10 min in waterbath.
- Take 10 µl, dilute in 990 µl dH2O in
cuvette and measure OD (600 nm) after 1 min (value should be 10%
of the value before zymolase treatment).
- Spin for 5 min at 800 rpm.
- Move to COLD ROOM (and keep everything on ICE!).
- Wash cells in 5 ml ice-cold spheroblast wash buffer.
- Spin for 5 min at 800 rpm, aspirate the supernatant (about 450
µl cell pellet remains).
- Resuspend in 450 µl EB buffer (gives 900 µl whole
cell extract) and transfer to 1.5 ml Eppendorf.
- Add 20 µl 10 % Triton X-100, vortex and leave on ice for
3 min.
- In the meantime, prepare 900 µl EBX-S in a 2 ml Eppendorf.
- Slowly lay 900 µl of whole cell extract onto EBX-S.
- Spin for 10 min at 12000 rpm.
- Take off yellow supernatant above the sucrose cushion (= extract
of soluble proteins).
- Pre-clear supernatant with 100 µl protein A-sepharose
(Amersham, equilibrated overnight in EBX buffer) for 30 min on
a rotating wheel.
- Spin for 1 min at 800 rpm.
- Transfer supernatant into 1.5 ml Eppendorf and add 2 µl
12CA5 (anti-HA) or 9E10 (anti-Myc) antibody.
- Leave on ice for 1 h.
- Add 20 µl protein A-sepharose.
- Allow binding reaction for 30 min on rotating wheel.
- Wash protein A-sepharose 8 times with 1 ml EBX.
- Add 40 µl 2X sample solution.
- Vortex and then boil 5 min at 95° C.
- Spin for 1 min at 13000 rpm.
- Transfer supernatent into new 1.5 ml Eppendorf.
- Load 20 µl on gel for silver staining.
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