2 Materials
All solutions have to be prepared using ultrapure water and analytical grade reagents. Unless otherwise stated, all reagents are
prepared at room temperature. All waste disposal regulations are
to be carefully followed upon the discarding of used materials and
reagents.
2.1 Buffers
for Chromoplast
Isolation
1. Buffer A: 100 mM Tris–HCl, pH 8.2, 330 mM sorbitol, 2 mM
MgCl 2 , 10 mM KCl, 8 mM EDTA, 10 mM L-ascorbic acid,
5 mM L-cysteine, 0.2% BSA. This buffer can be stored at 4
C.
Before use add DTT to 1 mM final concentration and PVPP to
1% (see Note 1).
2. Buffer B: Same as buffer A, but without PVPP.
3. Sucrose solutions: 15%, 30%, 40%, and 50% (w/v) solutions
prepared in 50 mM Tris–HCl (pH 7.4). These solutions can be
stored at À20
C. Add DTT to 1 mM final concentration
before use (see Note 2).
4. 1 M DTT stock solution in water. Aliquots can be stored at
À20
C (stable for up to 1 year).
2.2 Buffers
for Chromoplast
Fractionation
1. Lysis buffer: 50 mM HEPES, pH 8.0, 5 mM MgCl 2 , 17%
(w/v) sucrose. The stock can be stored at À20
C for up to
6 months. Add DTT to 1 mM final concentration immediately
before use.
2. Sucrose solutions (to be prepared immediately before use): 0%,
7%, 20%, 30%, 40% and 50% (w/v) solutions in 50 mM HEPES
pH 8.0, 5 mM MgCl 2 . These solutions can be stored at
À20
C. Add DTT to 1 mM final concentration (see Note 3).
3. PMSF protease inhibitor: 1 M solution in DMSO. Aliquots can
be stored for up to 6 months at À20
C.
3 Methods
Keep all reagents and samples on ice and carry out all procedures at
4
C. Short handling of centrifuge tubes and samples outside ice or
a cold room is acceptable.
3.1 Chromoplast
Isolation
1. Cut about 1 kg of tomato fruits into halves and remove seeds
and the rest of the gelatinous material of the locular cavities (see
Note 4).
2. Cut fruits into small pieces and place in a cold Waring blender
jar. Add approx. 2 volumes of buffer A per weight of tomato
tissue (see Note 5) and homogenize at low speed (three pulses
lasting for 1 s each once full speed is attained).
Chromoplast Fractionation
191
All solutions have to be prepared using ultrapure water and analytical grade reagents. Unless otherwise stated, all reagents are
prepared at room temperature. All waste disposal regulations are
to be carefully followed upon the discarding of used materials and
reagents.
2.1 Buffers
for Chromoplast
Isolation
1. Buffer A: 100 mM Tris–HCl, pH 8.2, 330 mM sorbitol, 2 mM
MgCl 2 , 10 mM KCl, 8 mM EDTA, 10 mM L-ascorbic acid,
5 mM L-cysteine, 0.2% BSA. This buffer can be stored at 4
C.
Before use add DTT to 1 mM final concentration and PVPP to
1% (see Note 1).
2. Buffer B: Same as buffer A, but without PVPP.
3. Sucrose solutions: 15%, 30%, 40%, and 50% (w/v) solutions
prepared in 50 mM Tris–HCl (pH 7.4). These solutions can be
stored at À20
C. Add DTT to 1 mM final concentration
before use (see Note 2).
4. 1 M DTT stock solution in water. Aliquots can be stored at
À20
C (stable for up to 1 year).
2.2 Buffers
for Chromoplast
Fractionation
1. Lysis buffer: 50 mM HEPES, pH 8.0, 5 mM MgCl 2 , 17%
(w/v) sucrose. The stock can be stored at À20
C for up to
6 months. Add DTT to 1 mM final concentration immediately
before use.
2. Sucrose solutions (to be prepared immediately before use): 0%,
7%, 20%, 30%, 40% and 50% (w/v) solutions in 50 mM HEPES
pH 8.0, 5 mM MgCl 2 . These solutions can be stored at
À20
C. Add DTT to 1 mM final concentration (see Note 3).
3. PMSF protease inhibitor: 1 M solution in DMSO. Aliquots can
be stored for up to 6 months at À20
C.
3 Methods
Keep all reagents and samples on ice and carry out all procedures at
4
C. Short handling of centrifuge tubes and samples outside ice or
a cold room is acceptable.
3.1 Chromoplast
Isolation
1. Cut about 1 kg of tomato fruits into halves and remove seeds
and the rest of the gelatinous material of the locular cavities (see
Note 4).
2. Cut fruits into small pieces and place in a cold Waring blender
jar. Add approx. 2 volumes of buffer A per weight of tomato
tissue (see Note 5) and homogenize at low speed (three pulses
lasting for 1 s each once full speed is attained).
Chromoplast Fractionation
191