Protocol for fractionation of Yeast (S1/S2)
Davis Lab
1.
Grow strains in rich or selective media to OD600 = 0.5.
2.
Harvest by centrifugation at 4200 X g .
3.
Wash twice with ddH
2
O.
4.
Weigh pellet; resuspend in 2ml/g TS buffer.
5.
Incubate 15 min. at 30°C.
6.
Wash once with HS buffer.
7.
Disrupt cell pellet by vortexing.
8.
Generate spheroplasts by incubation in 5ml/g of HS + 0.2mg/ml oxalyticase at
30°C.
9.
When 70% of cells are spheroplasted, stop reaction by placing cells on ice and gently adding 10ml/g PSM buffer.
10.
After 5 min. on ice, lyse by adding lysis buffer to a concentration of 20 OD600 units/ml, then incubate for 5 min. on ice.
11.
Collect soluble supernatant (S1) by centrifugation at 12,000 X g for 45 min at
4°C, then freeze aliquots in liquid nitrogen.
12.
Resuspend pellet in S2 buffer at 100 OD600 units/ml.
13.
Incubate on ice for 10 min.
14.
Spin at 12,000 X g for 45 min. at 4°C. Freeze aliquots in liquid nitrogen.
15.
Resuspend pellets in Laemmli buffer at a concentration of 100 OD600 units/ml; freeze in liquid nitrogen.
Buffers :
TS buffer :
 100mM Tris·SO
4
pH 9.4
 10mM DTT
 AqPI
HS buffer
 20mM Hepes pH 7.4
 1.2M sorbitol
 AqPI
PSM buffer
 20mM PIPES pH 6.5

 1mM MgCl

Lysis buffer


1.2M sorbitol
AqPI
2
20mM Hepes, pH 7.4
 5mM MgCl
AqPI
2

To make S1's: (No EDTA is present in this protocol since EDTA affects the nucleotide exchange of Ran).
1.
Grow 500ml yeast cell culture to OD600 = 0.6 (~300 OD units of cells).
2.
Spin cells down in GS3 rotor at 5,000 rpm for 5 min. in 50ml conical tubes.
3.
Weigh pellet (~1g/500ml cells -- depends on whether cells are grown in rich YPD or minimal media).
4.
Add 2ml of Tris-DTT + Protease Inhibitors per gram of cells. Incubate at 30˚C for
15 min. and pellet cells at 2,000 rpm for 5 min. at 4˚C.
5.
Wash cells with 5ml Sorbitol-Hepes + PI's buffer per gram of cells. Pellet as in
Step 4.
6.
Resuspend with 5ml/gram Sorbitol-Hepes+PI's plus 0.2mg lyticase/gram of cells.
7.
Incubate at 30˚C until spheroplasted and then place tube on ice. Spheroplasting can be monitored using the spectrophotometer. Take an initial OD600 using 980µl
0.1%SDS plus 20µl of cells. Take initial OD (wait about 10 sec. and record number). Check the drop in OD over the course of 30-45 min. You want to see
OD drop to about 1/10 of initial OD, representing about 90% cells spheroplasted.
8.
Slowly add 10ml of ice cold Sorbitol/Pipes/MgCl
2
/AqPI solution per gram of cells.
9.
Ice for 5 min. and gently pellet at 1,000 rpm at 4˚C (takes 10-15 min.).
10.
Aspirate supernatant well; vortex briefly, and then resuspend in 10ml/gram cold
Hepes/MgCl
2
/PI; this solution acts as a hypotonic solution which allows the cells to swell, and is added while vortexing.
11.
Tube is placed on ice for 10 min. as soon as cells are resuspended. While waiting, transfer cells to chilled 15ml Corex tube.
12.
Remove an aliquot of whole cell 5 OD of cells (5 X 200µl) and freeze immediately in liquid nitrogen; place in –80°C. Check lysis under microscope.
13.
When finished, add NaCl to cells while vortexing to a final concentration of
200mM.
14.
Spin cells for 45 min. at 9,000 rpm in SA600 at 4˚C. Remove supernatant aliquots
[prepare S1= 500µl aliquots using all the supernatant. Do not discard supernatant.
1g = 330 OD 10ml/gram = 33 OD/ml or 16.5 OD/500µl].
15.
Freeze in liquid nitrogen and store at –80°C. You do not need to move on to make
S2s but can to remove nucleoporins if you wish.
Fractions:
 Whole: freeze 5 x 200µl = ~6 OD each
 S1: freeze 5 x 800µl = ~24 OD each
 S2: freeze 10 x 1ml = ~30 OD each

Making S2s:
1.
Resuspend pellet from Step 10 in 10ml/gram Hepes/MgCl
2
/NaCl/AqPI solution.
2.
Vortex and ice for 10 min.
3.
Spin at 9,000 rpm for 45 min. at 4˚C.
4.
Remove supernatant aliquots [S2= 10 x 1ml ~33 OD each] and freeze in liquid nitrogen. Store at –80°C.
Buffers :
200X PI cocktail - prepare fresh!
 250µl 1000X Aprotinin/Leupeptin
 250µl 1000X TPCK
 250µl 1000X Pepstatin
 500µl 500X PMSF
Notes: Aprotinin is made as 1mg/ml in ddH
2
O and leupeptin as 0.5mg/ml in ddH
2
O; stable for 6 months stored at -20˚C . TPCK is made at 5mg/ml in ethanol and stable at room temperature.PMSF is made at 20mg/ml in isopropanol and stable at room temperature for 9 months.Pepstatin is made at 0.7mg/ml in MeOH and stored at -20˚C.
Tris/DTT (5ml)
 500µl 1M Tris·SO
4
(100mM final)
pH9.6
 50µl 1M DTT – add fresh!
(10mM final)
 4.45ml ddH
2
O
HEPES/MgCl


2
(20ml)
 400µl 1M HEPES pH7.4 (20mM final)
100µl 1M MgCl
19.5ml ddH
2
O
2
(5mM final)
Sorbitol/HEPES
 10ml 2.4M Sorbitol (1.2M final)
 400µl 1M Hepes pH 7.4 (20mM final)
(20ml)
 9.6ml ddH
2
O
Sorbitol/PIPES/MgCl
2
(20ml)
 10ml 2.5M Sorbitol (1.2M final)
 800µl 0.5M PIPES pH6.5
(20mM final)
 20µl 1M MgCl
2
(1mM final)
 9.2ml ddH
2
O
HEPES/MgCl
2
/NaCl (20ml)
 400µl 1M HEPES pH7.4 (20mM final)
 100µl 1M MgCl
2
(5mM final)
 4ml 5M NaCl (1M final)
 15.5ml ddH
2
O
Alternate S2 buffer





5mM MgCl
1mM DTT
0.5M NaCl
2
:
20mM Hepes pH7.4
5mM EDTA pH8 (replace with
for IPs)
0.5% Triton-X 100

 Protease Inhibitor cocktail

General Immunoprecipitation (IP) protocol
Davis lab 11/97
Preparing S1 fractions:
1.
Obtain yeast S1 fractions as per protocol above; bring to a final concentration of:
 50mM Hepes pH7
 200mM NaCl
 5mM MgCl2
 0.1% Tween 20
 +Protease inhibitors
 +/- GTP (use 0.2mM final concentration) using 2X compensating buffer. Use 250µl S1/sample + 250µl compensating buffer.
2.
Spin in TL100 10 min. at 50,000 rpm to clear any precipitate.
Preparing beads:
3.
Measure out 50µl of 20% Protein A Sepharose beads (PAS) [=10µl packed beads].
4.
Add 5µl antibody (antiserum or ascites fluid).
5.
Add 500µl compensating buffer (concentration is same as above).
6.
Incubate 1 hr. at 4˚C.
7.
Wash 2X with same buffer.
8.
Add diluted extracts to beads.
9.
Rotate 2 hr. at 4˚C.
10.
Wash 6X with ice cold buffer.
11.
Add 2X sample buffer containing 100mM DTT.