Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 42)

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2-D Electrophoresis Guide Methods Chapter 8: Sample Preparation
Cool protein precipitation and wash
solutions to –20°C and chill a mortar
with liquid nitrogen.
Place leaves in the mortar, add liquid
nitrogen, and grind the leaves
in the liquid nitrogen to a fine powder.
Transfer leaf powder into 20 ml protein
precipitation solution and incubate for
1 hr at –20°C. Stir solution occasionally.
Centrifuge the solution at –20°C for
15 min at 35,000 × g.
Discard the supernatant, add wash
solution, and suspend the pellet.
Incubate for 15 min at –20°C and stir
the solution occasionally.
Repeat steps 4 and 5 until the wash
solution turns from dark to light green.
Invert the column sharply several
times to resuspend the settled gel
and remove any bubbles. Snap off
the tip and place the column in a
2.0 ml microcentrifuge tube (included).
Remove the top cap. If the column does
not begin to flow, push the cap back on
the column and then remove it again to
start the flow. Allow the excess packing
buffer to drain by gravity to the top of
the gel bed (about 2 min). Discard the
drained buffer, then place the column
back into the 2.0 ml tube.
Centrifuge for 2 min in a microcentrifuge
at 1,000 × g to remove the remaining
packing buffer. Discard the buffer.
Apply the new buffer in 500 μl aliquots.
After each application, let the buffer
drain out by gravity, then centrifuge
the column at 1,000 × g for 1 min
to remove the buffer. Discard the
buffer from the collection tube.
Repeat as required. Three washes
result in >99% of the buffer exchanged.
Four washes result in >99.9% of the
buffer exchanged.
Centrifuge the solution at –20°C for
15 min at 35,000 × g and discard
the supernatant.
Add 2 ml of wash solution and suspend
the pellet.
Transfer the suspension into a shallow
ceramic shell and cover with perforated
Parafilm wrap.
Put the shell into a dessicator and
apply a vacuum until the pellet
(acetone powder) is dry.
Mix 5 mg of sample powder with
~0.5 ml of 2-D sample solution
and incubate for 30 min at room
temperature. Vortex from time to time.
Centrifuge the solution at room
temperature for 15 min at >16,000 × g.
Collect the supernatant and perform
the protein assay.
Place the column in a clean 1.5 or 2.0 ml
microcentrifuge tube. Carefully apply
the sample (20–75 μl) directly to the
center of the column. Application of
more or less than the recommended
sample volume may decrease
column performance.
Centrifuge the column for 2–4 min at
1,000 × g. Following centrifugation,
the purified sample is in the new
buffer. Molecules smaller than the
column’s exclusion limit are retained
by the column.
Plant Leaves
Plant leaf cells contain reactive compounds (such as proteases, phenol oxidases, organic acids, phenols,
and terpenes). To minimize the deleterious effects of these compounds on protein integrity, use the MicroRotofor
cell lysis kit (plant) or follow this protocol, which involves grinding the tissue in a mortar and pestle with liquid
nitrogen. Precipitate the proteins with 20% trichloroacetic acid (TCA) in prechilled acetone (–20°C). To remove
the plant phenols, rinse the pellet at least twice with cold acetone (–20°C) and air-dry samples in a vacuum
(Damerval 1986).
Reagents
■ ■
Protein precipitation solution
■ ■
Wash solution
■ ■
2-D sample solution
Protocol
Sample Cleanup
Prior to IEF, remove contaminating salts, buffers, and other chemicals from samples by dialysis, precipitation,
or buffer exchange. A protocol for buffer exchange using Bio-Rad’s Micro Bio-Spin
™ P-6 columns is provided
here. Another alternative is the use of the ReadyPrep 2-D cleanup kit to effectively precipitate sample protein
and remove contaminants. It has the additional benefit of concentrating the sample to a desired volume.
Buffer Exchange (Desalting)
Bio-Rad’s Micro Bio-Spin columns are suitable for use with 1.5 or 2.0 ml microcentrifuge tubes and are
completely autoclavable. They accommodate volumes of 20–75 µl; volumes less than 20 µl may affect
recovery. The gel in the Micro Bio-Spin columns is suspended in either SSC buffer, pH 7.0, or Tris-HCl buffer,
pH 7.4. For 2-D electrophoresis, it is best to exchange the sample into the 2-D sample solution (7 M urea,
2 M thiourea, 4% CHAPS) using the following protocol. DTT and ampholytes are added after the buffer
exchange procedure.
Protocol
1 1 7 4
3
9
5
4
5
6
10
2
11
3
12
13
2 8
Cell Lysis and Protein Extraction Procedures
(contd.)