Biology Reference
In-Depth Information
is the same as that of 1D-immnoblotting, but double-staining of
all protein spots and IgE-binding spots is adapted for precise local-
ization of the IgE-binding spots. From our experience,
14 cm × 14 cm 2D-gels seems to be the optimal size for handling
and obtaining good performance [
11
,
12
,
18
,
24
].
2
Materials
Prepare all solutions using ultrapure water and analytical grade
reagents.
Protect your eyes and hands when handling human serum, to
prevent potential transmission of infections. Wearing gloves
throughout the experiments is recommended for avoiding protein
contamination.
2.1 Sample
Preparation
1. Extraction buffer: 0.1 M phosphate-buffered saline (PBS,
pH 7.2), 0.1 M Tris-HCl, pH 7.0, 1 M NaCl is frequently
used (
see
Note 1
).
2. 2-D Clean-Up Kit (GE Healthcare UK Ltd., Little Chalfont,
UK): Store Wash buffer in the kit should be stored at −30 °C.
3. DeStreak Rehydration Solution (GE Healthcare): Before use,
equilibrate at RT and dissolve urea crystals, and then add 0.5 %
of IPG buffer and mix well.
4. Cellulose acetate 0.45
m fi lter (Dismic-45, Advantec Toyo
Kaisha Ltd., Tokyo, Japan).
5. Pierce BCA Assay Kit (Thermo Fisher Scientifi c K.K.,
Yokohama, Japan) or 2D-Quant Kit (GE Healthcare).
μ
1. Immobiline DryStrip pH3-10NL, 13 cm (GE Healthcare).
2. DryStrip holder, 13 cm (GE Healthcare).
3. IPG buffer pH3-10NL (GE Healthcare).
4. IPG phor III (GE Healthcare).
5. Cover oil fl uid (GE Healthcare).
6. Equilibration buffer: 50 mM Tris-HCl, pH 8.8, 6 M urea,
30 % (v/v) glycerol, 2 % (w/v) SDS, and a small amount of
bromophenol blue (BPB). Weigh 72.07 g of urea and 4 g
of SDS and add 10 mL of 1 M Tris-HCl (pH 8.8), 69 mL of
glycerol, and a small amount of BPB. Bring up the volume up
to 200 mL and stir at room temperature (RT). Divide into
aliquots and store at −20 °C.
7. Dithiothreitol: Just before use, add 100 mg (1 %) to 10 mL of
equilibration buffer.
8. Iodoacetamide: Just before use, add 250 mg (2.5 %) to 10 mL
of equilibration buffer.
2.2 Isoelectro-
phoresis and Sodium
Dodecyl Sulfate-
Polyacrylamide Gel
Electrophoresis
(SDS-PAGE)
