Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 41)

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2-D Electrophoresis Guide Methods Chapter 8: Sample Preparation
Cell Lysis and Protein Extraction Procedures
Pellet the cells by centrifugation at
2,000 × g for 5 min at 4°C.
Discard the supernatant and wash
pelleted cells in cold cell washing buffer.
Repeat steps 1 and 2 two times.
Add 2-D sample solution to the
pelleted cells and suspend the pellet
with a pipet.
Place the cell suspension on ice,
incubate 5 min, and sonicate at
appropriate intervals. Check lysis
efficacy by light microscopy.
Centrifuge cell debris at 14,000 × g
for 15 min and transfer supernatant
to a new vial.
Perform a protein assay of the
supernatant. A protein concentration
of 3–5 mg/ml is best for
2-D electrophoresis.
Chill a mortar with liquid nitrogen,
then grind small tissue pieces in
the presence of liquid nitrogen to
a fine powder.
Immediately after grinding, transfer
60 mg tissue powder to a
microcentrifuge tube containing
1.0 ml of 2-D sample solution.
Optional: sonicate the sample on
ice 5 times, for 2 sec each time.
Pause between sonication steps
to avoid overheating.
Incubate the sample at room
temperature for 30 min. Vortex from
time to time.
Centrifuge at 35,000 × g for 30 min
at room temperature.
Perform a protein assay to determine
the protein concentration of the
supernatant, which should be
5–10 mg/ml.
Dilute the supernatant with 2-D
sample solution and incubate for
20 min at room temperature.
Carefully remove (decant) culture
medium from cells. Wash cells twice
with cell washing buffer.
Add 2-D sample solution to the cells
and keep on ice for 5 min. Swirl the
plate occasionally to spread the buffer
around the plate.
Use a cell scraper to collect the lysate
and transfer to a microcentrifuge tube.
Place the cell suspension on ice,
incubate 5 min, and sonicate at
appropriate intervals. Check lysis
efficacy by light microscopy.
Centrifuge the cell debris at 14,000 × g
for 15 min and transfer the supernatant
to a new vial.
Perform a protein assay of the
supernatant. A protein concentration
of 3–5 mg/ml is best for
2-D electrophoresis.
Centrifuge cells (~5 × 10 7 ) at 5,000 × g
for 3 min and resuspend the pellet in an
equal volume of 2-D cell washing buffer
heated at 37°C and centrifuge again.
Repeat two more times to remove
all interfering material (extracellular
proteases and growth media).
Add ~150 µl hot (95°C) SDS sample
solubilization buffer to the pellet and
vortex thoroughly.
Sonicate the sample solution 10 times
for 1 sec each at ~60 W and ~20 kHz.
Incubate the sample at 95°C for 5 min.
Cool the sample to 20°C and dilute
with ~500 µl of 2-D sample solution.
Incubate for another 20 min at room
temperature. The final SDS concentration
should not exceed 0.25% in the extract
to be applied onto the IPG strip; therefore,
be sure that the total volume is
maintained during the SDS boiling step.
Centrifuge the sample solution at 20°C
for 30 min at 14,000 × g and harvest
the supernatant.
Perform the protein assay. The protein
concentration should be ~5 µg/µl.
Suspension Cultured Human Cells
Use the MicroRotofor ™ cell lysis kit (mammalian) or the
protocol below, which uses 2-D sample solution and a
sonicator for cell lysis and protein extraction. Use 0.5 ml
of 2-D sample solution with 3 × 10
7 cells.
Reagents
■ ■
2-D sample solution
■ ■
Cell washing buffer
Protocol
Mammalian Tissue
Use the MicroRotofor cell lysis kit (mammalian) or the
protocol below, which involves freezing tissue samples
(for example, biopsy samples) in liquid nitrogen.
Use liquid nitrogen and a mortar and pestle to grind
the samples while they are still frozen. Break up any
larger pieces beforehand (for example, wrap the
frozen tissue sample in aluminum foil and crush
with a hammer).
Reagents
■ ■
2-D sample solution
Protocol
Monolayer Cultured Human Cells
Use the MicroRotofor cell lysis kit (mammalian) or
the protocol below, which uses 2-D sample solution
and a sonicator for cell lysis and protein extraction.
Use 0.5 ml of 2-D sample solution with 3 × 10
7 cells.
Reagents
■ ■
2-D sample solution
■ ■
Cell washing buffer
Protocol
Microbial Cultures
Reproducible sample preparation from bacteria and
yeast is challenging because the cells may release
proteases and other enzymes into the growth medium
(Harder et al. 1999, Drews et al. 2004, Poetsch and
Wolters 2008). Wash the cultures thoroughly with isotonic
buffers and take precautions to inactivate the proteolytic
activity after cell lysis. Extensive disruption of microbial
cells is required and is usually performed with the help of
a French press, bead impact instruments, or sonicator.
Use the MicroRotofor cell lysis kit (bacteria),
the MicroRotofor cell lysis kit (yeast), or the protocol
below. This protocol relies on cell lysis with ultrasonic
waves in combination with a solubilization in SDS
under elevated temperature to ensure deactivation
and denaturation of proteases.
Reagents
■ ■
SDS sample solubilization buffer
■ ■
2-D sample solution
■ ■
Cell washing buffer
Protocol
1
1
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3
3
3
3
4
4
4
4
5
5
5
5
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7
6
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7
2
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