Biology Reference
In-Depth Information
1. Add mitochondrial protein sample (10-100
μ
g protein) to
3.4.1 Marker Enzyme
Assays of Mitochondria
L reaction master mix.
2. Add malate to start reaction to a fi nal concentration of 50 mM.
3. Progression of the reaction is measured directly at 340 nM
(
900
μ
Fumarase
ε
= 2.55 mM
−1
).
1. Add mitochondrial protein sample (10-100
μ
g protein) to
Aconitase
L reaction master mix.
2. Add aconitate to start reaction to a fi nal concentration of
8 mM (
see
Note 23
).
3. Progression of the reaction is measured as NADP reduction to
NADPH at 340 nM (
900
μ
ε
= 6.22 mM
−1
).
1. Pipette 0.5 mL 2× reaction buffer and mitochondrial protein
sample (10-100
3.4.2 Marker Enzyme
Assay of Chloroplast
Contamination
g protein).
2. 2× reaction buffer: 200 mM Tris-HCl (pH 7.8), 20 mM
MgCl
2
, 40 mM KCI, and 20 mM DTT.
3. Add 2 mM ATP, 2 mM phosphoenol pyruvate, 0.2 mM
NADH, 3.5 U/mL pyruvate kinase, and 5 U/mL lactate
dehydrogenase.
4. Add H
2
O to 975
μ
Phosphoribulokinase
L and mix.
5. Monitor the
A
340nm
until constant.
6. Perform a baseline correction.
7. Add 25
μ
L 20 mM ribulose-5-phosphate (Ru5P) (fi nal con-
centration 0.5 mM) to initiate the reaction.
8. Activities are assayed by following the oxidation of NADH at
340 nm (
μ
ε
= 6.22 mM
−1
).
1. Catalase activity can be measured in an oxygen electrode,
by monitoring the production of oxygen after the addition
of H
2
O
2
.
2. Set up the electrode according to the manufacturer's instruc-
tions using 50 % (w/v) saturated KCl as an electrolyte and
calibrate between air-saturated water (253 nmol O
2
/mL at
25 °C) and zero (established by adding a few crystals of sodium
hydrosulfi te to the water) (
see
Note 21
).
3. The assay should be conducted at 25 °C. To 1 mL of mito-
chondrial reaction medium add 5-20
3.4.3 Marker Enzyme
Assay of Peroxisome
Contamination
Catalase
μ
L of mitochondrial sus-
pension (
see
Note 22
).
4. Add 4
L of 1 % (v/v) H
2
O
2
and monitor oxygen evolution.
Rates of oxygen evolution should be calculated as nmol O
2
/
min mg protein. Typical rate for highly purifi ed peroxisomes is
~14
μ
μ
mol O
2
/min mg protein.
