Microsome Isolation Yeast (Pompon et al)

FOR pYEDP60 vector:

Day 1 (afternoon): inoculate 50 ml culture SGI medium with yeast colony in 250 ml flask.
Grow at 28 C – 250 rpm
Day 2: -
Day 3 (afternoon): Centrifuge culture in bluecap 5 min 1000g (2300 rpm table centrifuge)
Resuspend pellet in 250 ml YPL medium
Grow at 28 C - 250 rpm o/n
Day 4 (morning): measure OD600 – YPL medium as blanc (about 6 is OK)
Microsome isolation

Centrifuge culture 4 min 7000 rpm

Resuspend pellet in 25 ml TEK-buffer
Leave 5 min at room temperature
Centrifuge culture 4 min 7000 rpm
Resuspend pellet in 2,5 ml TES-B (transfer to bluecap, keep on ice)
Add glassbeads up to level of cell suspension (Wear gloves, work in fumehood!!!)
Shake by hand 6x 1 minute (so total 6 minutes). Keep on ice inbetween shaking.
Add 5 ml TES-B buffer -> mix
Remove 5 ml (up to glass beads), transfer to 30 ml centrifuge tube.
Add 5 ml TES-B buffer -> mix
Remove 5 ml (up to glass beads), transfer to the same 30 ml centrifuge tube.
Add 5 ml TES-B buffer -> mix
Remove 5 ml (up to glass beads), transfer to the same 30 ml centrifuge tube.
Centrifuge final volume of 15 ml – 10 min 15000 rpm at 4 C.
Remove supernatant to fresh tube (note: pellet is not very tight, not able to recover complete 15 ml)
(When sup not clear, could transfer to fresh tube and repeat centrifugation)
Add new TES-B up to 15 ml
Split into 2 new 30 ml centrifuge tubes (2 x 7.5 ml)
To each tube add 6.5 ml TES-B
To precipitate microsomes add to each tube: 2 ml TES-B-NaCl and 4 ml TES-B-PEG
(final concentration NaCl = 0.15 M and PEG 4000 = 0.1 g/ml)
Mix
Leave at least 15 minutes on ice
Centrifuge 10 minutes 10000 rpm at 4 C
Resuspend the 2 pellets from 1 isolation into 3 ml TEG.
Use a 5 ml potter to homegenize the pellet. Keep on ice at all times.
Tranfer microsomes to bluecap. Add 15 ul 1M DTT (to get a final concentration of 5mM DTT)
200 ul Aliquots in eppendorf tubes (pre-chilled)
Store microsomes at -70 C.
10X YNB: (13.4% Yeast Nitrogen Base with Ammonium Sulfate without amino acids)
Dissolve 134 g of yeast nitrogen base (YNB) in 1000 ml of MQ.
If necessary warm up the solution to dissolve YNB completely in water.
Filter sterilize. Store at +4°C.

50% Glucose: Dissolve 25 g D-Glucose / 50 ml MQ

Filter sterilize

50% Galactose: Dissolve 25 g D-Galactose / 50 ml MQ

Filter sterilize

SGI: 1 g/l Bacto casaminoacids

20 mg/l Tryptophan (I)
6.7 g/l Yeast Nitrogen Base (without aa with ammoniumsulfate)
20 g/l Glucose

400 ml: 0,4 g casaminoacids

8 mg Tryptophan
Up to 364 ml MQ ------> 2 x 250 ml bottle 182 ml per bottle
Autoclave
After autoclave add: 10 ml 10X YNB
8 ml 50 % Glucose

YPL: 10 g/l Yeast Extract

10 g/l Bactopeptone
20 g/l Galactose

1000 ml: 10 g Bactopeptone

10 g Yeast extract
Up to 960 ml MQ ------> 4 x 250 ml bottle = 240 ml per bottle
Autoclave
After autoclave add: 10 ml 50% Galactose

Glass beads: 425-600 microns (Sigma G-9268)

Wash beads subsequently with: Soap
Water
70 % EtOH
Water
Autoclave beads in water
Poor off water, dry beads in 65 C stove
Ready to use
TEK buffer (25 ml / isolation needed): 50 mM TrisHCl pH 7.4
1 mM EDTA
0.1 M KCl (mw 74,56)
250 ml: 12.5 ml 1M TrisHCl 7.4
0.5 ml 0.5 M EDTA
1.864 g KCl
Up to 250 ml MQ
Autoclave

TES-B buffer (56,5 ml / isolation needed): 50 mM TrisHCl pH 7.4

1 mM EDTA
0.6 M Sorbitol (mw 182,17)
500 ml: 25 ml 1M TrisHCl 7.4
1 ml 0.5 M EDTA
54.651 g sorbitol
Up to 500 ml MQ
Autoclave

TES-B NaCl buffer (4 ml / isolation needed): 50 mM TrisHCl pH 7.4

1 mM EDTA
0.6 M Sorbitol
1.5 M NaCl (mw 58.44)
100 ml: 5 ml 1 M TrisHCl 7.4
0.2 ml 0.5 M EDTA
10.93 g sorbitol
8.766 g NaCl
Up to 100 ml MQ
Autoclave

TES-B PEG buffer (8 ml / isolation needed): 50 mM TrisHCl pH 7.4

1 mM EDTA
0.6 M Sorbitol
0.5 g/ml PEG 4000
50 ml: dissolve 25 g PEG 4000 in about 15 ml MQ (use microwave)
dissolve 5,47 g sorbitol in about 10 ml MQ (use microwave)
Add together + 2.5 ml 1 M TrisHCl 7.4
0.1 ml 0.5 M EDTA
Up to 50 ml MQ
Autoclave

TEG buffer (3 ml / isolation needed): 50 mM TrisHCl pH 7.4

1 mM EDTA
20 % glycerol
100 ml: 5 ml 1 M TrisHCl pH 7.4
0.2 ml 0.5 M EDTA
20 ml glycerol
Up to 100 ml MQ
Autoclave