Biomedical Engineering Reference
In-Depth Information
2
Materials
1. 10× TE: 100 mM Tris-HCl, 10 mM ethylenediamine-
tetraacetic acid (EDTA), pH 7.5. Store at room temperature.
Working concentration is 1×.
2. 10× LiAc solution: 1 M LiOAc, pH 7.5 (adjusted with
acetic acid). Store at room temperature. Working concen-
tration is 1×.
3. 50 % polyethylene glycol (PEG): 50 g PEG-4000 in
100 mL. Store at room temperature. Working concentration
is 40 %.
4. Salmon sperm DNA: 10
2.1 Transformation/
Transfection
2.1.1 Yeast
Transformation
μ
g/
μ
L, store in small aliquots at
−20 °C.
5. Vectors:
see
Fig.
1
.
1. Lipofectamine™ 2000 (Invitrogen).
2. Opti-MEM
®
I Reduced Serum Medium (Gibco).
2.1.2 Mammalian Cell
Transfection
2.2 Verifi cation
of Expression
1. 2 % SDS-containing protease inhibitors: add one tablet prote-
ase inhibitors (cOmplete, EDTA-free [Roche]) to 25 mL of
2 % sodium dodecyl sulfate (SDS) in water, and store in ali-
quots at −20 °C.
2. Acid-washed glass beads 0.45 mm diameter (Sigma).
3. Laemmli sample buffer: 0.3 M Tris-HCl (pH 6.8), 30 % glyc-
erol, 10 % SDS, 1.54 M dithiothreitol (DTT), 0.4 mg/mL
bromophenol blue. Store in 1 mL aliquots at −20 °C.
2.2.1 Lysate Preparation
Yeast Lysate Preparation
1. Wash: PSB (phosphate buffered saline).
2. Lysis buffer: 10 mM HEPES, pH 7.6, 150 mM NaCl, 0.5 mM
MgCl
2
, 10 % glycerol, 0.5 % Triton X-100, 0.5 % sodium
deoxycholate, protease inhibitor cocktail [cOmplete, EDTA-
free (Roche)].
3. Laemmli sample buffer (
see
Subheading “Yeast Lysate
Preparation,”
item 3
, of Subheading
2.2.1
).
Mammalian Lysate
Preparation
1. 12 % separating gel: 4.65 mL distilled water, 1.25 mL 3 M
Tris-HCl (pH 8.8), 0.05 mL 20 % SDS, 4 mL acrylamide/bis-
acrylamide (30 %/0.8 %), 0.05 mL 10 % ammonium persulfate
(APS), 0.005 mL tetramethyl-ethylenediamine (TEMED).
Mix directly before use. This amount is suffi cient for preparing
two gels with a spacer thickness of 0.75 mm when using a GE
Healthcare gel running system.
2. 4 % stacking gel: 4.12 mL distilled water, 0.21 mL 3 M Tris-
HCl (pH 6.8), 0.025 mL 20 % SDS, 0.67 mL acrylamide/bis-
acrylamide (30 %/0.8 %), 0.025 mL 10 % APS, 0.005 mL
2.2.2 SDS-PAGE
