Biology Reference
In-Depth Information
4. 100 mM phospho(enol)pyruvic acid monosodium salt hydrate
in 1× reaction buffer.
5. 10 mM NADH in 1× reaction buffer, prepare only on the day
of use.
6. Pyruvate kinase (e.g., rabbit muscle), 400-800 U/mg protein.
7. Lactate dehydrogenase (e.g.,
Lactobacillus leichmannii
), 150-
300 U/mg protein.
8. H
2
O.
9. Visible wavelength spectrophotometer.
1. Clark-type electrode, such as those manufactured by Hansatech
(Kings Lynn, UK) or Rank Brothers (Bottisham, UK).
2. Reaction medium: 10 mM TES-KOH, pH 7.2, 0.3 M sucrose,
5 mM KH
2
PO
4
, 10 mM NaCl, 2 mM MgSO
4
, and 0.1 %
(w/v) BSA.
3. Sodium hydrosulfi te.
4. 1 % (v/v) H
2
O
2
solution.
5. Mitochondria isolated according to Subheading
3.2
.
2.4.3 Marker Enzyme
Assay of Peroxisome
Contamination
Catalase
There are two main gel-based methods to separate the plant mito-
chondrial proteins for DIGE analysis (Fig.
1
). Blue-native (BN)
SDS-PAGE DIGE is well suited to profi le the composition of indi-
vidual ETC complexes, and detailed procedures of BN-SDS-PAGE
[
48
] and BN-SDS-PAGE-DIGE [
49
] are available and are not
covered here. 2D-IEF-SDS-PAGE is a highly reproducible plat-
form that is commonly used for mitochondrial proteomic analysis,
and the combination of DIGE and 2D-IEF-SDS-PAGE (2D-IEF-
SDS-PAGE-DIGE) provides a powerful tool to defi ne contami-
nants when comparing two mitochondrial isolation procedures.
2.4.4 Differential In-Gel
Electrophoresis of Purifi ed
Mitochondrial Proteins
1. Acetone.
2. Microcentrifuge such as Eppendorf 5430 or an equivalent.
3. Lysis buffer 1: 8 M Urea, 4 % (w/v) CHAPS, 40 mM Tris
(2-amino-2-hydroxymethyl-propane-1,3-diol) (
see
Note 10
).
Mitochondrial Precipitation
and Resuspension
1. Cy-Dyes Cy5, Cy3, and Cy2 (GE Healthcare Life Sciences).
2. Dimethylformamide (DMF) (water-free).
3. 10 mM Lysine.
4. Vortex and microfuge.
5. Lysis buffer 2: 8 M Urea, 4 % (w/v) CHAPS, 40 mM Tris
(2-amino-2-hydroxymethyl-propane-1,3-diol), 72 mM DTT
(
see
Note 10
).
CyDye Labelling
