Plant Mitochondrial Proteomics - Plant Proteomics: Methods and Protocols - page 483

3. Mitochondrial reaction medium: 0.3 M mannitol, 10 mM
TES-KOH (pH 7.5), 3 mM MgSO 4 ·7H 2 O, 10 mM NaCl,
5 mM KH 2 PO 4 , 0.1 % (w/v) BSA.
4. Stock solutions of respiratory substrates: 100 mM NADH;
1 M succinate; 50 mM ATP; 500 mM pyruvate; 50 mM
malate; 30 mM NAD + ; 10 mM thiamine pyrophosphate;
12 mM coenzyme A. Coenzyme A should be made up in 1 %
(w/v) cysteine and all other components should be made up
in 500 mM TES-KOH, pH 7.5.
5. 100 mM Adenosine 5 -diphosphate (ADP) made up in

500 mM TES-KOH, pH 7.5.

′

1. Visible wavelength spectrophotometer.
2. 0.1 M KH 2 PO 4 -NaOH, pH 7.7.
3. 10 % (v/v) Triton X-100.
4. 1 M Malate.
5. Mitochondria isolated according to Subheading 3.2 .
6. Reaction master mix: 70 mM KH 2 PO 4 -NaOH, pH 7.7,
0.05 % (v/v) Triton X-100. 900

2.4 Determining
Mitochondria Purity

2.4.1 Marker Enzyme
Assays of Mitochondria

Fumarase

L master mix per assay (i.e.,
to fi ll a 1 mL spectrophotometric cuvette after subsequent
additions of sample and substrate) ( see Note 9 ).

μ

1. Visible wavelength spectrophotometer.
2. 0.1 M HEPES-NaOH, pH 7.5.
3. 10 % (v/v) Triton X-100.
4. 20 mM Nicotinamide adenine dinucleotide phosphate
(NADP).
5. 0.1 M MnCl 2 .
6. 2,000 U/mL NADP-isocitrate dehydrogenase (ICDH) (por-
cine heart).
7. 0.2 M Aconitate.
8. Mitochondria isolated according to Subheading 3.2 .
9. Reaction master mix: 80 mM HEPES-NaOH, pH 7.5, 0.05 %
(v/v) Triton X-100, 0.5 mM NADP, 0.5 M MnCl 2 , 2 U
NADP-ICDH. 900

Aconitase

L master mix per assay (i.e., to fi ll a 1 mL
spectrophotometric cuvette after subsequent additions of sam-
ple and substrate) ( see Note 9 ).

μ

1. Mitochondria isolated according to Subheading 3.2 .
2. 2× reaction buffer: 200 mM Tris-HCl (pH 7.8), 20 mM
MgCl 2 , 40 mM KCI, 20 mM DTT.
3. 100 mM adenosine 5

2.4.2 Marker Enzyme
Assay of Chloroplast
Contamination

′ -triphosphate (ATP) disodium salt in 1×

Phosphoribulokinase

reaction buffer.