Biomedical Engineering Reference
In-Depth Information
1. Cold-saturated ammonium sulfate, pH 7.2; store at 4 °C, stable
for years.
2.4.1 Import of Urea-
Denatured Precursor
Proteins
2. Urea buffer: 7 M urea, 30 mM MOPS-KOH, pH 7.2, 1 mM
DTT (
see
Note 1
), prepare fresh.
2.5 Analysis
of the Import Reaction
1. Standard materials required to perform SDS-PAGE and
Western blot.
2. Highly specifi c antiserum against DHFR (or against other
utilized protein tags).
3. Quantifi cation of Western blot signals by image analysis software
(i. e. ImageJ).
2.6 Dissection
of a Functional Import
Intermediate on Blue
Native PAGE
1. Gel preparation and running settings for Blue Native PAGE.
(a) ATP-regenerating system: creatine kinase, 10 mg/ml, pre-
pare fresh; creatine phosphate, store as 1 M stock at
−20 °C, stable for years.
(b) 5 % digitonin in water, prepare a 5 % stock solution in
water (
see
Note 4
), keep at 4 °C, stable for months.
(c) Solubilization buffer: 1 % digitonin, 20 mM Tris-HCl,
pH 7.4, 0.1 mM EDTA, 50 mM NaCl, 10 % glycerol,
1 mM PMSF (
see
Note 4
). Prepare a 2× solution w/o
digitonin, and add detergent prior to use.
(d) Acrylamide solution: 49.5 % Acrylamide (e.g.,
Roth
),
1.5 % bis-acrylamide, store at room temperature in the
dark, stable for months.
(e) 3× gel buffer: 200 mM
ʵ
-amino-
n
-caproic acid, 150 mM
bis-tris/HCl, pH 7.0, store at 4 °C, stable for months.
(f) 10× Loading Dye: 5 % Coomassie blue G250 (water solu-
ble, whereas Coomassie R is not), 500 mM
-amino-n-
caproic acid, 100 mM bis-tris/HCl, pH 7.0. Prepare
10 ml and store at 4 °C, stable for months.
(g) 10× anode buffer: 500 mM bis-tris/HCl, pH 7.0, store at
4 °C, stable for months; 1× solution can be used for up to
three times.
(h) 10× cathode buffer: 500 mM tricine, 150 mM bis-tris/
HCl, pH 7.0, prepare 1 L, divide into halves, and dissolve
0.2 % Coomassie blue G250 in 500 ml.
(i) Marker protein set (e.g.,
Sigma-Aldrich
).
(j) Gradient mixer, peristaltic pump, cooled vertical gel elec-
trophoresis system (e.g., SE 600 Series,
GE Healthcare
).
ʵ
