Biology Reference
In-Depth Information
Rabbit antibodies against Erg6p, Wbp1p, Cyb2p, GAPDH from
S. cerevisiae
Peroxidase-conjugated secondary antibody
Ammonium carbonate (NH
4
CO
3
)
Dithiothreitol (DTT)
Iodoacetamide
Trypsin
Trifluoroacetic acid (TFA)
Alpha-cyano-4-hydroxycinnamic acid
[Glu
1
]-Fibrinopeptide B
Solvents: acetic acid, acetone, acetonitrile, chloroform, diethyl ether, formic
acid, light petroleum, methanol
Washing solutions for lipid extracts: 0.034% MgCl
2
; 2 N KCl/MeOH (4:1; v/v);
artificial upper phase (chloroform/methanol/water, 3:48:47; per vol.)
Charring solution: 0.63 g MnCl
2
4H
2
O, 60 mL water, 60 mL methanol, 4 mL
conc. H
2
SO
4
ANSA solution: 40 g K
2
S
2
O
5
, 0.63 g of 8-anilino-1-naphthalenesulfonic acid and
1.25 g of Na
2
SO
3
in 250 mL of water
2.1.3
Databases
MASCOT Database (
http://www.matrixscience.com
)
Saccharomyces
Genome Database (
http://www.yeastgenome.org
)
Swissprot Protein Database (
http://www.uniprot.org/
)
2.2
METHODS
2.2.1
Isolation of LD from yeast
LD from
S. cerevisiae
are isolated from 4 to 5 L of full or selective media. Cultures
are inoculated from a preculture to an OD
600
of 0.1 and cells are grown to the sta-
tionary phase at 30
C with shaking. Yeast cells are harvested by centrifugation at
5000 rpm for 5 min at room temperature (RT) and washed with distilled water
(see Note 1). After determining the cell wet weight cells are incubated with
0.5 g/mL SP-A (0.1 M Tris/SO
4
[pH 9.4]) and 1.54 mg DTT/mL SP-A for 10 min
at 30
C with shaking. Then, cells are washed once in prewarmed SP-B (1.2 M sorbi-
tol, 20 mM KH
2
PO
4
[pH 7.4]) and spheroplasts are generated by enzymatic digestion
of the cell wall using Zymolyase-20 T (Seikaguku Corporation) at a concentration
of 2 mg/g cell wet weight in 6 mL SP-B/g cell wet weight. The incubation takes
30 min-1 h at 30
C with shaking. The resulting spheroplasts are washed twice with
cold SP-B. From now on, cells must be kept on ice and all solutions must be precooled.
Spheroplasts are resuspended in 1 mL/g cell wet weight LD-A (12% Ficoll 400
in 10 mM MES/Tris [pH 6.9], 0.2 mM Na
2
EDTA
2H
2
O) and 1 mM PMSF followed
by mechanical disruption with 30 strokes using a Dounce homogenizer with a loose
fitting pestle. The resulting homogenate is diluted with a half volume of LD-A
