Bio-Rad GS-900™ Calibrated Densitometer - Manual (Page 40)

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2-D Electrophoresis Guide Methods Chapter 8: Sample Preparation
■ ■
Direct application of clarified lysate to IPG strips
is appropriate only for samples with high protein
content and minimal interfering substances.
Preparation of many sample types (for example,
plant tissues and dilute bodily fluids) should
incorporate a precipitation step to remove
interfering substances and allow application
of a more concentrated sample
Protein Solubilization
■ ■
Prepare fresh sample solubilization solutions
daily or store them frozen in aliquots, preferably
at –80°C; always use high-quality reagents and
proteomics-grade water. Use urea stock solutions
soon after they are made, or treat them with a
mixed-bed ion exchange resin to avoid protein
carbamylation by cyanate, which forms in old urea.
If solutions are prepared in advance and stored,
it is best to prepare them without reductant (DTT)
and add the reductant directly before use
■ ■
Dissolve pelleted protein samples in 1×
2-D sample solution
■ ■
Perform a protein quantitation assay to determine
the amount of total protein in each sample. Use a
protein assay that is tolerant to chemicals in your
samples. For samples in 2-D sample solution,
for example, use the RC DC
™ protein assay,
which can tolerate up to 2% detergent
■ ■
Dilute or concentrate samples as needed to
yield a final protein concentration of 1–5 mg/ml.
Make dilutions in 2-D sample solution and
concentrate the sample using the ReadyPrep
™
2-D cleanup kit
■ ■
Use protein extracts immediately or aliquot them
into appropriately sized batches and store them
at –80°C to avoid freeze-thaw cycles
■ ■
Highly viscous samples likely have a very high
DNA or carbohydrate content. Fragment DNA
with ultrasound during protein solubilization or by
adding endonucleases like benzonase. Use protein
precipitation (for example, with the ReadyPrep 2-D
cleanup kit) to diminish carbohydrate content
■ ■
Do not heat samples containing urea and thiourea
above 35°C as this can lead to protein modification
Tips for Sample Preparation
Keep the sample preparation workflow simple
(increasing the number of sample handling steps
may increase variability).
Lysis (Cell Disruption)
■ ■
For each 10 mg (fresh weight) pelleted cells or
animal tissue, use about 1 ml of 2-D sample
solution for a protein concentration of 1–3 mg/ml.
When disrupted in liquid nitrogen, samples such
as liver biopsies and plant leaves contain 10–30%
and 1–2% extractable protein, respectively
■ ■
To diminish endogenous enzymatic activity:
— Disrupt the sample or place freshly disrupted
samples in solutions containing strong
denaturing agents such as 7–9 M urea,
2 M thiourea, or 2% SDS. In this environment,
enzymatic activity is often negligible
— Perform cell disruption at low temperatures
to diminish enzymatic activity
— Lyse samples at pH >9 using either sodium
carbonate or Tris as a base in the lysis solution
(proteases are often least active at basic pH)
— Add a chemical protease inhibitor
to the lysis buffer. Examples include
phenylmethylsulfonyl fluoride (PMSF),
aminoethyl-benzene sulfonyl fluoride (AEBSF),
tosyl lysine chloromethylketone (TLCK),
tosyl phenyl chloromethyletone (TPCK),
ethylenediaminetetraacetic acid (EDTA),
benzamidine, and peptide protease inhibitors
(for example, leupeptin, pepstatin, aprotinin,
bestatin). For best results, use a combination
of inhibitors in a protease inhibitor cocktail
— If protein phosphorylation is to be studied,
include phosphatase inhibitors such as fluoride
and vanadate
■ ■
When working with a new sample, use at least two
different cell disruption protocols and compare
the protein yield (by protein assay) and qualitative
protein content (by SDS-PAGE)
■ ■
Optimize the power settings of mechanical
rupture systems and incubation times for all
lysis approaches. Mechanical cell lysis usually
generates heat, so employ cooling where required
to avoid overheating of the sample
■ ■
Following cell disruption, check the efficacy of
cell wall disruption by light microscopy and
centrifuge all extracts extensively (20,000 × g
for 15 min at 15°C) to remove any insoluble
material; solid particles may block the pores
of the electrophoresis gel
2-D sample solution (50 ml)
7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 40 mM DTT,
0.2% (w/v) ampholytes (pH 3–10)
Urea/thiourea stock solution
48 ml
CHAPS 2.0 g
Bio-Lyte ® ampholytes, pH 3–10 250 µl
DTT 0.31 g
Bromophenol blue (1%) 10 µl
Distilled or deionized H
2
O to 50 ml
■ ■
2-D sample solution is used for sample application
and IPG strip rehydration. Bio-Rad offers various
types of 2-D sample buffers, which differ in
solubilizing power (see Ordering Information)
■ ■
For pH control, Tris base may be added to the
2-D sample solution at 10–40 mM. Addition of
Tris increases the conductivity of the sample solution
and extends the time required to focus the IPG strips
■ ■
Ampholytes are added to all IPG rehydration
and sample solubilization solutions to maintain
solubility of the proteins. The choice of ampholytes
depends on the pH range of the IPG strip.
Higher concentrations (up to 1% (w/v)) may be
used, but they result in lower IEF voltage and
correspondingly longer focusing times
Urea/thiourea stock solution (50 ml)
Urea 22 g
Thiourea 8 g
Distilled or deionized H
2
O to 50 ml
Filter through Whatman No. 1 paper
using a Buchner funnel
Store at –80°C
1% Bromophenol blue (10 ml)
Bromophenol blue will not dissolve in unbuffered
water. Prepare 10 ml of 50 mM Tris base by dissolving
60.6 mg of Tris in 10 ml of water. Add 100 mg of
bromophenol blue and vortex until dissolved.
Store at 25°C.
Cell washing buffer (1 L)
10 mM Tris-HCl, pH 7.0, 250 mM sucrose
Tris base
1.21 g
Sucrose 85.58 g
Distilled or deionized H
2
O 800 ml
Dissolve
Adjust pH to 7.0 with HCl
Distilled or deionized H
2
O to 1 L
Store at 4°C
Protein precipitation solution (100 ml)
20% (w/v) trichloroacetic acid (TCA), 0.2% DTT (w/v)
in ice-cold acetone (–20°C)
Trichloroacetic acid
20 g
DTT 0.2 g
Acetone 80 ml
Dissolve
Acetone
to 100 ml
Store at –20°C
Wash solution (100 ml)
0.2% DTT in ice-cold acetone (–20°C)
DTT
0.2 g
Acetone 80 ml
Dissolve
Acetone
to 100 ml
Store at –20°C
SDS sample solubilization buffer (50 ml)
1% (w/v) SDS, 100 mM Tris-HCl (pH 9.5)
SDS
0.5 g
Tris base 0.6 g
Distilled or deionized H
2
O 40 ml
Titrate to pH 9.5 with diluted HCl
Distilled or deionized H
2
O to 50 ml
Store at 25°C
Buffers and Solutions