Biology Reference
In-Depth Information
3. Transfer the 27 mL sample (6-11 mg protein per tube) in a
centrifuge tube (50 mL) and underlay it carefully with 8 mL of
the gradient medium, using a peristaltic pump (4 mL/min).
Centrifuge at 10,000 ×
g
for 3 h at 4 °C in a swing-out rotor.
4. The opaque tonoplast enriched band is localized between the
sucrose cushion and the supernatant (Fig.
1
). It is transfered
carefully into a centrifugation tube and fi lled up to 35 mL with
dilution medium and centrifuged at 50,000 ×
g
for 30 min at
4 °C. The pellet on the bottom of the tube presents the intra-
cellular membranes (Endoplasmic reticulum, Golgi, micro-
bodies, etc.), which is resuspended in dilution medium and
centrifuged similar to the tonoplast sample.
5. The fi nal pellets are resuspended in the storage medium and
stored below −70 °C until use.
3.5 Protein
Quantifi cation
1. Add
x
μ
L of the sample corresponding to 2-10
μ
g of protein to
a 3 mL cuvette.
2. Add dilutor up to 500
L and mix well.
3. Add 2.5 mL Bradford reagent, mix and wait for 5-10 min.
4. Measure with the spectrophotometer at 595 nm and compare
to a standard curve prepared with BSA (2-12
μ
μ
g of protein)
(
see
Note 8
).
The different protein fractions are fi rst isolated as explained in
Subheadings
3.1
to
3.4
and followed by modSDS-PAGE or native
electrophoretic techniques (hrCNE and native IEF). The used elec-
trophoretic methods are soft enough to allow the detection of per-
oxidases with different staining methods.
All methods have in common that no reducing agents are used
and samples are not heated. The modSDS-PAGE, which is also
applied for the second dimension, contains 0.1 % SDS in all buffer
systems. However, SDS concentrations up to 1 % are possible for
the detection of peroxidases. The enzymes are still specifi cally
detectable by this procedure as they stay intact. The two other elec-
trophoretic methods, IEF and hrCNE, do work without SDS.
3.5.1
Electrophoresis
The modSDS-PAGE is used for the fi rst dimension as well as for the
second dimension after IEF and hrCNE. For the modSDS-PAGE
in the fi rst dimension 1 M urea is added to the solutions for a better
resolution, whereas no urea is used for the second dimension. To
get a better resolution of the proteins modSDS-PAGE is casted as a
gradient gel: 4-18 % for fi rst dimension or second dimension after
IEF and 4-16 % for second dimension after hrCNE.
3.6 Preparation of
Polyacrylamide Slab
Gels
3.6.1 Modifi ed SDS-Gel
1. Prepare 4 and 16 % acrylamide resolving gel mixture for sec-
ond dimension after hrCNE (Subheading
2.10
) or 4 and 18 %
acrylamide resolving gel mixture for second dimension after
