Biology Reference
In-Depth Information
a
b
unwashed
washed
pH 3
10
S
M
PM
T
M
PM
T
130
95
72
55
43
34
36
17
Fig.
2
ModSDS-PAGE as fi rst and second dimension. (
a
) Peroxidases were separated by modSDS-PAGE
(4-18 %) with 1 M urea for soluble (S), microsomal fractions (M), plasma membrane (PM) and tonoplast (T).
On each lane 20
g total protein were loaded. Membrane fractions were used either unwashed or washed with
150 mM KCl in the presence of 0.01 % Triton X-100. (
b
) 2D-PAGE separation of soluble peroxidases (20
μ
g
protein) in native IEF-PAGE, pH 3-10 followed by second dimension modSDS-PAGE (4-18 %). On the
left
prestained marker (Fermentas, Germany) is shown with corresponding molecular weight in kD. Peroxidases
were stained with DAB (
a
,
b
)
μ
3. Resuspend the membranes in 20
L IEF-LB (for native IEF
and modSDS-PAGE) or hrCNE-LB (both 1× concentration).
4. Add digitonin to the hrCNE samples in the protein to deter-
gent ratio of 1:5.
5. Solubilize samples for 1 h on ice with interim mixing.
6. Centrifuge samples at 16,000 ×
g
for 60 min at 4 °C. Load
supernatant for IEF and hrCNE on the corresponding gel. For
modSDS-PAGE supernatant is desalted, using ZebaTM Spin
Desalting Columns, 7K MWCO (Thermo Scientifi c, Germany).
Desalted samples are mixed 1:1 with (2× SDS-LB).
7. Soluble proteins (20
μ
g) can be directly mixed with the sample
buffers and loaded on the gel.
μ
3.8 Electrophoresis
1. Put the gel system together. Fill both chambers with running
gel buffer.
2. Transfer the samples (Subheading
3.7
) in the gel pockets. Add
one marker lane (Fig.
2
).
3. Electrophoresis is carried out with 30 mA per gel up to 200 V at
4 °C until the blue front reaches the bottom of the gel.
3.8.1 ModSDS-PAGE
