Biology Reference
In-Depth Information
5. After centrifugation, the mitochondria should form a yellow-
brown band toward the top of the tube. Collect this band with
a Pasteur pipette and dilute with at least 4 volumes of WS
without BSA, and centrifuge at 21,000 × g for 15 min at 4 °C
in 50 mL tubes.
6. The resulting loose pellet is resuspended in WS without BSA
and centrifuged again at 21,000 × g for 15 min. The mito-
chondrial pellet is resuspended in a small volume of WS
(~1 mL) without BSA.
7. This will yield a concentration of ~5-20 mg mitochondrial
protein/mL ( see Note 20 ).
3.3 Determining
Mitochondria Quality
Respiratory rates of isolated mitochondria can be measured as
substrate-dependent oxygen consumption using an oxygen elec-
trode. The dependence of respiration rate upon added ADP can be
used to calculate the respiratory control ratio that gives an indica-
tion of the extent to which the mitochondria are coupled/intact.
1. 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 ).
2. The assay should be conducted at 25 °C. To 1 mL of mito-
chondrial reaction medium add 5-20
g of mito-
chondrial protein) of mitochondrial suspension ( see Note 22 ).
Determine the background rate of oxygen consumption [rate
( a )]. Add one of the following substrate combinations (all
concentrations are fi nal), 1 mM NADH; 10 mM succinate and
0.5 mM ATP; 5 mM pyruvate, 0.5 mM malate, 0.3 mM
NAD + , 0.1 mM thiamine pyrophosphate and 12
μ
L (~100
μ
M CoA and
determine the rate of oxygen consumption. Add 1
μ
L of
100 mM ADP. The rate of oxygen consumption should
increase: measure the initial linear rate [rate ( b )]. After a few
minutes the ADP will be depleted and the rate of oxygen con-
sumption will be reduced to [rate ( c )]. The respiratory control
ratio is given by
μ
[
rate
( )
c
rate
( )]
a
RCR
=
[
rate
( )
b
rate
( )]
a
During the isolation of mitochondria most contamination occurs
as co-purifi cation of similarly dense chloroplasts and peroxisomes.
By profi ling the activity of certain enzymes that are known to be
localized in these other cellular compartments, we can determine
the purity of isolated mitochondria.
3.4 Determining
Mitochondrial Purity
Search WWH ::




Custom Search