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12
Proteomics
Sample Preparation
30 g
leaves
Cut leaves
into pieces
Proteomics
Sample Preparation
SAMPLE PREPARATION
Chloroplast Isolation Kit
For the isolation of intact chloroplasts from plant leaves
Transfer supernatant
to fresh tubes
Add Chloroplast
Isolation Buffer
1x CIB
with BSA
Blend or
homogenize
Remove
supernatant
Centrifuge
1000 x g for 7 min.
Chloroplasts
appear as
green pellet
Resuspend
each pellet
in 1x CIB
with BSA
Pass through
filter mesh;
collect liquid
Layer the
chloroplast
suspension on
a Percoll gradient
or layer
Divide filtrate into 4,
50 ml tubes
Discard
pellets
Centrifuge
200 x g for 3 min.
Centrifuge
to separate broken
chloroplasts from intact
chloroplasts
(1-6 min. depending
on plant type)
This kit enables isolation of chloroplasts through mechanical cell wall and cell
membrane breakage, removal of cell debris and unbroken leaf tissue by filtration,
collection of total cell chloroplast fraction by centrifugation, and separation of
intact chloroplasts from broken chloroplasts using a Percoll® layer or gradient.
Sigma’s Chloroplast Isolation Kit has been tested for use with spinach, pea, lettuce,
cabbage, mangold and tobacco.
Features & Benefits
• Specifically formulated buffer – Saves time using easy-to-follow, simplified
protocol based on proven methods
• Isolates intact chloroplasts – Useful for studies of such processes as carbon
assimilation, electron flow and phosphorylation, metabolic transport, or protein
targeting
• Kits are useful for both proteomic or genomic applications – Provides
optimized conditions for isolation of intact chloroplasts from leaves of plants
commonly used for research
Components
Chloroplast Isolation Buffer 5x (CIB)
Percoll®
Ferricyanide dependent oxygen evolution by
chloroplasts – a measure of intact chloroplasts
This assay is based upon the inability of the ferricyanide
(artificial electron acceptor) to cross the chloroplast envelope and react with the electron transport system in the
intact thylakoid membranes. Electron transport from water
to ferricyanide results in oxygen release. The degree of
integrity of the chloroplast preparation is assessed by comparing the rates of oxygen evolution upon illumination
before and after osmotic shock of the chloroplasts.
Bovine Serum Albumin (BSA)
Filter Mesh 100
Ferricyanide photoreduction – a measure of intact chloroplasts
This assay is based upon the inability of the ferricyanide to cross the chloroplast envelope and to react with the electron
transport system in the thylakoid membranes. Ferricyanide reduction, as indicated by the decrease in the absorbancy at 410
nm, occurs only when ruptured chloroplasts are present in the preparation.
The degree of integrity of the chloroplast preparation is assessed by comparing the rate of ferricyanide reduction upon
illumination before and after osmotic shock of the chloroplasts.
250
3.2
0.04
50
0
Lettuce intact 93%
Chloroplasts (equivalent to 30 µg/ml chlorophyll) prepared
using CP-ISO were illuminated in the presence of 1.5 mM
ferricyanide and 5 µmole NH4Cl, at 25 °C. Evolution of oxygen was measured by an oxygen electrode. The blue bars
represent the level of oxygen evolved by the chloroplast
preparation in isotonic medium indicating the fraction of
ruptured chloroplasts within the preparation, while the
violet bars represent the level of oxygen evolved by the
same chloroplast preparation after osmotic shock (ruptured
chloroplasts). Calculation of the correlation between the
oxygen evolution before and after the osmotic shock indicates that the spinach and the lettuce chloroplast preparations contain 89% and 93% intact chloroplasts, respectively.
Slope ( OD410/min)
3
150
Spinach intact 89%
s i g m a - a l d r i c h . c o m
A
B
200
OD410
mole O2/mg chlorophyll
Analysis of the results, presented here, indicates that 88% of the chloroplasts in the spinach chloroplast preparation are intact.
300
before
after
2.8
2.6
2.4
0.03
0.02
0.01
0
0
2
Time/Min
4
6
before
after
Chloroplasts (equivalent to 25 µg/ml chlorophyll) prepared using CP-ISO were illuminated in the presence of 1.5 mM
ferricyanide. The reduction of ferricyanide was measured spectrophotometrically (410 nm).
Graph A demonstrates the change in absorbancy of the two samples (before and after osmotic shock) during 6 minutes.
Graph B shows bars representing the slopes of the lines in Graph A.
Product Code
Description
Size
CP-ISO
Chloroplast Isolation Kit
1 kit
13
SAMPLE PREPARATION
Proteomics
Sample Preparation
Mitochondria Isolation Kit
For the isolation of an enriched, functional mitochondrial fraction from
animal tissue
This kit features specially formulated extraction reagents and optimized protocols
for the preparation of mitochondrial fractions from either soft (liver or brain) or
hard (skeletal or heart muscle) tissue.
In addition, the integrity of the inner membrane of the resulting mitochondria is
analyzed by measuring the uptake of the fluorescent carbocyanine dye (JC-1) which
is supplied in the kit. The integrity of the mitochondrial outer membrane may be
assessed by measuring cytochrome c oxidase activity using the Cytochrome c
Oxidase Assay Kit (CYTOC-OX1). The resulting intact mitochondrial preparation is
suitable for apoptosis studies, 2D analysis for proteomics and many other applications. Each kit is sufficient for the extraction of mitochondria from 10 to 20 grams
of animal tissue. Enough JC-1 staining solution is provided to perform 50 JC-1
assays of 2 ml each.
Features & Benefits
• Specially formulated extraction reagents and a proven
procedure suitable for small scale isolation – Isolate an
enriched, intact mitochodrial fraction in a microfuge tube
• Produces functionally active, intact mitochondria – Resulting
mitochondria are suitable for in vitro assays for apoptosis,
oxidative stress or other studies
• Includes stain and protocol for determining inner
membrane integrity – Determine the integrity of the inner
mitochondrial membrane without the need to purchase
other reagents
• Compatible with the Cytochrome c Oxidase Assay Kit –
Allows easy determination of the integrity of the outer
mitochondrial membrane
Components
Extraction Buffer A, 5x
Extraction Buffer B, 5x
Storage Buffer, 5x
Albumin Solution
JC-1 Stain
JC-1 Assay Buffer, 5x
s i g m a - a l d r i c h . c o m
Trypsin
14
SAMPLE PREPARATION
Proteomics
Sample Preparation
Small Scale
[Large Scale (3-10 g tissue)
is also possible]
Mitochondria Isolation Kit Continued
Western blot of rat heart and liver mitochondria
Soft tissue
(brain or liver)
94
48.6
36.4
29.8
20.6
6.5
MW
Liver mitochondria
2.5 mgP/ml
Control
cytochrome c
50 g/ml
Heart mitochondria
2.5 mgP/ml
Another indication for the integrity of mitochondria is the presence of cytochrome c in the intermembrane space (between
the inner and outer membranes). The presence of free cytochrome c in whole mitochondria can be demonstrated by a
Western blot of the preparations produced using the MITO-ISO1 kit. Mitochondria from heart show more cytochrome c (at
the same total protein concentration) than mitochondria from liver.
Samples were analyzed by 20% SDS-PAGE and then blotted. Antibody specific to cytochrome c was then used in Western
blot analysis. Protein concentrations of the samples used are indicated at the bottom.
These results show that functional, intact mitochondria are prepared using the Mitochondria Isolation Kit. In addition, the use
of this kit together with the Cytochrome c Oxidase Assay Kit facilitates a quick and easy determination of the functionality of
both the inner and outer mitochondrial membranes.
Linearity of JC-1 uptake in mitochondria from various organs
Linearity of JC-1 uptake
vs amount of mitochondria
600
550
rat liver
rat brain
500
rat skeletal muscle
rat heart
mouse liver
450
Fluorescence
400
350
300
250
200
150
100
50
0
0
5
10
15
20
25
30
35 40
g mitochondrial protein
45
50
55
60
s i g m a - a l d r i c h . c o m
The cationic dye JC-1 (5,5’-6,6’-tetrachloro-1,1’-3,3’tetraethlybenzimidazolyl-carbocyanine iodide) can be used to measure
the mitochondrial inner membrane potential since it is actively taken up in respiring mitochondria. The uptake is energy
dependent and needs ATP to power the potential gradient formed over the inner mitochondrial membrane. The linear range
is from 5-30 µg mitochondrial protein for many tissue extracts.
Product Code
Description
Size
MITO-ISO1
Mitochondria Isolation Kit
1 kit
15
SAMPLE PREPARATION
Proteomics
Sample Preparation
Cytochrome c Oxidase Assay Kit
For the determination of cytochrome c oxidase activity in soluble and
membrane bound mitochondrial samples
Cytochrome c oxidase is located on the inner mitochondrial membrane dividing
the mitochondrial matrix from the intermembrane space. The Cytochrome c
Oxidase Kit uses an optimized colorimetric assay based on observation of the
decrease in absorbance of ferrocytochrome c measured at 550 nm, which is caused
by its oxidation to ferricytochrome c by cytochrome c oxidase. This kit is suitable for
the detection of mitochondrial outer membrane integrity and for the detection of
mitochondria in subcellular fractions.
The Cytochrome c Oxidase Kit provides sufficient reagent for 100 tests.
Features & Benefits
• Simple, optimized protocol – Obtain reproducible results without the need for
special training
• Useful for determination of cytochrome c activity from any mitochondrial
source – The enzyme is present in all mitochondria regardless of species
• Useful for detecting the presence of mitochondria in subcellular
fractions – Save time and increase confidence in the quality of organelle
preparations
• May be used in conjunction with the Mitochondrial Isolation
Kit (MITO-ISO1) – Standardized mitochondrial preparation and analysis ensure
reproducible results
Components
Assay Buffer 5x
Enzyme Dilution Buffer 2x
Cytochrome c
Sodium Hydrosulfite
Cytochrome c Oxidase (positive control)
n-Dodecyl b-D-Maltoside
Cytochrome c Oxidase Kit determines percentage of intact mitochondria
prepared with Mitochondria Isolation Kit
Integrity of mitochondria prepared from rat tissues
100
% intact mitochondria
% damaged mitochondria
90
80
Percent
70
60
50
40
30
20
10
0
Rat skeletal muscle
Tissue
Rat heart muscle
Cytochrome c Oxidase activity is measured in the presence and absence of the detergent 1 mM n-dodecyl
b-D-maltoside. The ratio of the two activities provides a measure of the integrity of the outer mitochondrial
membrane.
Product Code
Description
Size
CYTOC-OX1
Cytochrome c Oxidase Assay Kit
1 kit
s i g m a - a l d r i c h . c o m
Rat liver
16
SAMPLE PREPARATION
Proteomics
Sample Preparation
Individual Protease Inhibitors
Inhibitor
AEBSF
Aprotinin saline solution
Aprotinin, affinity purified
Bestatin, hydrochloride
Chymostatin
E-64
EDTA disodium salt dihydrate
EDTA, 0.5 M solution, disodium salt
EGTA
Leupeptin, hemisulfate
Leupeptin, TFA salt
Leupeptin hydrochloride
Pepstatin A
Pepstatin A, 90%
1, 10-Phenanthroline
Phosphoramidon
PMSF
s i g m a - a l d r i c h . c o m
See Protease Inhibitor Cocktails. p. 64
Product
Code
A 8456
A 6279
A 4529
B 8385
C 7268
E 3132
E 5134
E 7889
E 8145
L 2884
L 2023
L 9783
P 4265
P 5318
P 9375
R 7385
P 7626
Application
Inhibits serine proteases, such as trypsin and chymotrypsin
Inhibits serine proteases such as trypsin chymotrypsin, plasmin, trypsinogen urokinase and kallikrein.
Aprotinin inhibits human leukocyte elastase but not pancreatic elastase
Inhibits aminopeptidases, such as leucine aminopeptidase
Inhibits serine and cysteine proteases
Inhibits cysteine proteases such as calpain, papain, and cathepsin B
Inhibits metalloproteases; chelates; permeabilized Gram negative bacteria
Inhibits metalloproteases, chelates
Inhibits both serine and cysteine proteases, such as calpain, trypsin, papain, and cathepsin B
Inhibits acid proteases such as pepsin (human or porcine), renin, cathepsin D, chymosin (bovine rennin),
and protease B
Inhibits metalloproteases
Inhibits thermolysin and collagenase
Serine protease inhibitor