2D PAGE- complete protocol

Sample preparation (analytical gels)

Sample preparation and solubilization are crucial factors for the overall
performance of the 2-D PAGE technique. Protein complexes and aggregates
should be completely disrupted in order to avoid appearance of new spots
due to a partial protein solubilization. The SWISS-2DPAGE samples were
prepared as followed:

Human samples

CEC (Colon epithelial cell): Within a maximum of 30 min, a large right or

left colon tissue sample (5 x 7 cm) was prepared on ice in the operating
room, rinsed with cooled phosphate buffer saline, pH 7.2 to further remove
cell debris and blood and then immersed into an iced PBS solution
containing 3 mM EDTA, 50 µg/ml leupeptine, 0.2 mM PMSF and 0.8 mM
benzamidine. Crypts were scraped away from the basal membrane with a
scalpel. The tissue was then gently pressed through a mesh with a pore size
of 300 µm to separate epithelial cells from stroma and filtered through a
nylon mesh with 200 µm pores. After centrifugation at 350 g at 4o C for 10
min, the membranes were permeabilized in 70 % chilled ethanol and the
cells were shaken overnight at 4o C. After washing with PBS, the cells were
incubated with fluorescein-conjugated anticytokeratin antibodies (CAM 5.2)
for 45 min and then sorted by FACS. The pellet was mixed with 100 µl per
106 cells of a solution containing urea (8 M), CHAPS (4 % w/v), DTE (65
mM), Tris (40 mM) and a trace of bromophenol blue. Hundred µl of the
final diluted colon epithelial cell sample was loaded onto the IPG gel strip.

CSF (cerebrospinal fluid): An aliquot of 250 µl of human CSF was mixed

with 500 µl of ice cold acetone and centrifuged at 10000 g at 4o C for 10
minutes. The pellet was mixed with 10 µl of a solution containing SDS (10%
w/v) and DTE (2.3% w/v). The sample was heated to 95o C for 5 minutes
and then diluted to 60 µl with a solution containing urea (8 M), CHAPS (4%
w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. The
whole final diluted CSF sample (45 µg) was loaded on the first dimensional
separation.

ELC (erythroleukemia cell line): A monolayer culture of human ELC was

grown, rinsed trypsinized and washed as explained in the HEPG2 sample
preparation. A pellet of 0.8 x 106 cells were mixed and solubilized with 60
µl of a solution containing urea (8 M), CHAPS (4% w/v), Tris (40 mM),
DTE (65 mM) and a trace of bromophenol blue. The whole final diluted
ELC sample was loaded on the first dimensional separation.

HEPG2 (hepatoblastoma carcinoma derived cell line): A monolayer culture

of human hepatoblastoma carcinoma derived cell line was grown in
Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf
serum (FCS). Cells were rinsed once with DMEM without FCS and
removed from the flask by incubating them with a solution containing
trypsin (0.5 g/l) and EDTA (0.2 g/l). After 3 minutes, DMEM containing
FCS was added into the flask to stop the action of the trypsin. The cells were
detached from the surface of the flask by squirting the solution onto the
cells. The suspension was transferred into a tube and the cells were
centrifuged at 1000 g during 5 minutes. Supernatant was discarded and the
cells were washed with DMEM without FCS. After centrifugation and
removal of DMEM, 0.8 x 106 cells were mixed and solubilized with 60 µl of
a solution containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65
mM) and a trace of bromophenol blue. The whole final diluted HEPG2
sample was loaded on the first dimensional separation.

HEPG2SP (hepatoblastoma carcinoma derived cell line secreted proteins):

Five ml of supernatant HEPG2 culture media were concentrated down to 30
µl in a MicrosepTM Concentrators. The concentrated sample was mixed
with 60 µl of a solution containing urea (8 M), CHAPS (4% w/v), Tris (40
mM), DTE (65 mM) and a trace of bromophenol blue. The whole final
diluted HEPG2SP sample was loaded on the first dimensional separation.

HL60 (promyelocytic leukemia cells): A monolayer culture of a human

promyelocytic leukemia cell line was grown in Dulbecco's modified Eagle
medium (DMEM) containing 10% fetal calf serum (FCS). Cells were rinsed
once with DMEM without FCS and removed from the flask by incubating
them with a solution containing trypsin (0.5 g/l) and EDTA (0.2 g/l). After 3
minutes, DMEM containing FCS was added into the flask to stop the action
of the trypsin. The cells were detached from the surface of the flask by
squirting the solution onto the cells. The suspension was transferred into a
tube and the cells were centrifuged at 1000 g during 5 minutes. Supernatant
was discarded and the cells were washed with DMEM without FCS. After
centrifugation and removal of DMEM, 0.8 x 106 cells were mixed and
solubilized with 60 µl of a solution containing urea (8 M), CHAPS (4%
w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. The
whole final diluted HL60 sample was loaded on the first dimensional
separation.

KIDNEY: Tissue resections were washed several times in cold rinse buffer
(glutamine-free RPMI 1640 medium containing 5 % fetal calf serum, 0.2
mM phenylmethylsulfonyl fluoridem, 1mM EDTA, and antibiotics:
oxacillin 25 µg/ml, gentamycin 50 µg/ml, penicillin 100 U/ml, streptomycin
100 µg/ml, amphotericin B 0.25 µg/ml, nistatin 50 U/ml) to further remove
cell debris and blood, and were frozen by immersion in liquid nitrogen. They
were then submitted to mechanical dissociation by scraping and gentle
squeezing. The cell suspension was washed several times in cold phosphate
buffer saline, pH 7.2. The cellular pellet was denatured with 100 µl per 106
cells of a solution containing urea (8 M), CHAPS (4 % w/v), DTE (65 mM),
Tris (40 mM) and a trace of bromophenol blue. Hundred µl of the final
diluted kidney cell sample was loaded onto the IPG gel strip.

LIVER Protocole 1: Five frozen slices (20 µm x 5 mm x 10 mm) of a human

liver biopsy were mixed with 300 µl of a solution containing urea (8 M),
CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol
blue. Sixty µl (45 µg) of the final diluted liver sample was loaded on the first
dimensional separation [2, 3].

LIVER Protocole 2: Ten mg of a human liver frozen biopsy was lyophillized

for 48 h. It was then crushed in a mortar containing liquid nitrogen and
mixed with 1.5 ml of a solution containing urea (8 M), CHAPS (4% w/v),
Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. Sixty µl (100
µg) of the final diluted liver sample was loaded on the first dimensional
separation.

LYMPHOMA: Five frozen slices (20 µm x 5 mm x 10 mm) of a human

lymphoma biopsy were mixed with 300 µl of a solution containing urea (8
M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of
bromophenol blue. Sixty µl (45 µg) of the final diluted lymphoma sample
was loaded on the first dimensional separation.

NUCLEI_LIVER_HUMAN: An aliquot of 106 nuclei was resuspended in 1

mL of sample buffer containing 5 M urea, 2 M thiourea, 2% w/v CHAPS,
2% w/v sulfobetain 3-10, 100 mM DTE, and 40 mM Tris. To inhibit the
interaction between nucleic acids and basic or nucleic acid binding proteins,
40 mM spermine and 0.01% w/v polyethyleneimine was added. After an
incubation period of 15 min at room temperature with several vortexing
steps, the sample was centrifuged at 15 000 x g for 10 min to pellet the
nucleic acid-polyamine complexes. After diluting 200 mL of the supernatant
to 450 mL using the same sample buffer, the carrier ampholyte
concentration was adjusted to a final concentration of 0.8% v/v before the
sample was laoded on the first dimensional separation.

PLASMA: An aliquot of 6.25 µl of human plasma was mixed with 10 µl of

a solution containing SDS (10% w/v) and DTE (2.3% w/v). The sample was
heated to 95o C for 5 minutes and then diluted to 500 µl with a solution
containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and
a trace of bromophenol blue. Sixty µl (45 µg) of the final diluted plasma
sample was loaded on the first dimensional separation [1].

PLATELET: Fresh blood has been centrifuged at 1000 g for 5 minutes. The
supernatant (platelet-rich plasma: RPR) was then centrifuged at 5000 g for
10 minutes. 106 washed platelets were suspended in 500 µl of lysis buffer
containing Tris-HCl pH 8.0 (10 mM), MgCl2 (1.5 mM), KCl (10 mM), DTE
(0.5 mM) and PMSF (0.5 mM) and incubated on ice for 10 minutes.
Mechanical lysis was achieved with a potter homogenizer and the resulting
lysate was centrifuged at 3000 g for 10 minutes. Then 1/10 volume of a
solution containing Tris-HCl pH 8.0 (0.3 M), KCl (1.4 M) and MgCl2 (30
mM) was added to the supernatant and ultracentrifuged at 54000 g for 2
hours. The supernatant was diluted with 3 volumes of distilled water to
decrease the salt concentration and then concentrated down to 30 µl in a
MicrosepTM Concentrator. The concentrated protein sample was mixed and
solubilized with 70 µl of a solution containing urea (8 M), CHAPS (4%
w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. The
whole final diluted platelet sample was loaded on the first dimensional
separation [5].

RBC (red blood cells): Fresh blood was centrifuged at 2500 g at 4o C for 10
minutes. Plasma and buffy coat were removed and RBC were washed three
times with the same volume of PBS pH 7.4. An aliquot of 7 µl of RBC was
mixed with 483 µl of a solution containing urea (8 M), CHAPS (4% w/v),
Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. Forty µl (45
µg) of the final diluted RBC sample was loaded on the first dimensional
separation [4].
U937 (macrophage like cell line): A monolayer culture of human U937 was
grown at a concentration of 0.5 million/ml in RPMI 1640 containing 1%
FCS. The cells were then rinsed, trypsinized and washed as explained in the
HEPG2 sample preparation. After centrifugation and removal of RPMI, 0.8
x 106 cells were mixed and solubilized with 60 µl of a solution containing
urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of
bromophenol blue. The whole final diluted U937 sample was loaded on the
first dimensional separation.

Mouse samples

BAT (Brown adipose tissue): Four hundred µg of dried brown adipose tissue
was mixed with 60 µl of a solution containing urea (8 M), CHAPS (4%
w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of
bromophenol blue. The whole final diluted sample (150 µg) was loaded in a
cup at the cathodic end of the IPG gels.

ISLETS (Pancreatic islet cells): Two hundred of small and large pancreatic
islets were mixed with 60 µl of a solution containing urea (8 M), CHAPS
(4% w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of
bromophenol blue. The whole final diluted sample (100 µg) was loaded in a
cup at the cathodic end of the IPG gels.

LIVER: Two hundred µg of dried liver was mixed with 60 µl of a solution

containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM),
SDS (0.05% w/v) and a trace of bromophenol blue. The whole final diluted
sample (100 µg) was loaded in a cup at the cathodic end of the IPG gels.

LIVER NUCLEI (Soluble nuclear proteins and matrix from liver tissue):
Nuclear proteins were solubilized in a buffer containing 5M urea, 2M
thiourea, 2% CHAPS (w/v), 2% sulfobetains (w/v), 65mM DTE, 40mM Tris
(pH 6.8), 0.8% Resolyte 3.5-10 and a trace of bromophenol blue.

MUSCLE (Gastrocnemius muscle): Two hundred µg of dried gastrocnemius

muscle was mixed with 60 µl of a solution containing urea (8 M), CHAPS
(4% w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of
bromophenol blue. The whole final diluted sample (100 µg) was loaded in a
cup at the cathodic end of the IPG gels.

WAT (White adipose tissue): Sixteen mg of dried white adipose tissue was
mixed with 60 µl of a solution containing urea (8 M), CHAPS (4% w/v),
Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of bromophenol
blue. The whole final diluted sample (150 µg) was loaded in a cup at the
cathodic end of the IPG gels.

Other samples

ARABIDOPSIS (Arabidopsis thaliana): Arabidopsis thaliana (L.) Heynh.

ecotype Columbia seeds were imbibed one hour in water and then sown in
soil (Teramax Belflot, Bachman SA, Chevroux, Switzerland) in growth
chambers under long-day condition (16/8 h light/dark cycle). The rosette
leaves were collected from 3 to 4 week-old plants and immediately frozen in
liquid nitrogen. The proteins were extracted according to Damerval et al.
[28], i.e. in acetone/ TCA mixture, and the extracts resuspended in 60 l. mg
-1 of 9M urea, 1% CHAPS, 1% DTT, 4% ampholines pH 3.5-10 and 1% pH
4.8-8 (BDH). Each strip was overnight rehydrated with 60 µl of the sample
(" in-gel sample rehydration "), and completed to a total of 450 µl with a
solution made of 9 M urea, 2% CHAPS, 15 mM DTT, 0.8% of ampholines
pH 3.5-10 (BDH) and a trace of Bromophenol blue.

DICTY (Dictyostelium discoideum): A WS380B wild type strain was used

here. Slugs (0.9 mg dry weight) were resuspended in 40 µl of a solution
containing urea (8 M), CHAPS (4 % w/v), Tris (40 mM), DTE (65 mM) and
a trace of bromophenol blue. Nine µl of this sample was diluted with 60 µl
of the same solution. This whole final Dicty diluted sample was loaded onto
the IPG gel strip.

ECOLI (Escherichia coli): Cells were grown aerobically in glucose minimal

morpholinopropane sulfonate (MOPS), plus thiamine at 37oC. Growth was
stopped in the late exponential phase at an OD of 1 at 600 nm. Five hundred
ml of culture medium was centrifuged for 30 min at 3000 rpm at 4\xa1 C
and the pellet was washed 4 times for 10 min at 4000 rpm in 10 ml low salt
washing sample buffer: KCl 3.0 mM, KH2PO4 1.5 mM, NaCl 68 mM,
NaH2PO4 9.0 mM. The pellet was then resuspended in 600 µl of a buffer
containing 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
DTE, 0.5 mM Pefabloc SC (protease inhibitor), 0.1% SDS, and stored at
-20o C. One µl of the latter was mixed with 60 µl of a solution containing
Urea (8 M), CHAPS (4 % w/v), Tris (40 mM), DTE (65 mM) and a trace of
bromophenol blue. After centrifugation at 10000 g for 5 minutes, the whole
final diluted E. coli sample was loaded onto the IPG gel strip.
YEAST (Saccharomyces cerevisiae): Cells were washed twice with PGSK
(NH2PO4-H2O 0.52 g/l, Na2HPO4-2H2O 8.8 g/l, NaCl 2.83 g/l, KCl 0.372
g/l and glucose 11 g/l), centrifuged at 3000 rpm for 5 min (4oC) and the
supernatant removed. The pellet was resuspended in 1 volume of PGSK and
1 volume of glass beads (425-600 µm diameter) and shaked for 10 min at
4oC in a bead beater. The extracts were centrifuged at 3000 rpm for 10 min
at 4oC, the supernatant retained and the pellet subjected these procedures a
second time. The supernatants were pooled and 100 µg (measured by
modified Lowry) of yeast proteins was mixed with 60 µl of a solution
containing urea (8 M), CHAPS (4 % w/v), Tris (40 mM), DTE (65 mM) and
a trace of bromophenol blue. The whole yeast diluted sample was loaded
onto the IPG gel strip.

Immobilized pH gradient (IPG) as first dimension

A non-linear immobilized pH gradient (3.5-10.0 NL IPG 18 cm) was used as

the first dimension. It offered high resolution, great reproducibility and
allowed high protein loads. Based on our specifications, the non-linear pH
gradient strips were prepared by Pharmacia-Hoeffer Biotechnology AB and
are commercially available. The strips were 3 mm wide and 180 mm long
[6-12].

IPG gel strips rehydration

Hydration was performed overnight in the Pharmacia reswelling cassette
with 25 ml of a solution containing urea (8 M), CHAPS (2% w/v), DTE (10
mM), Resolyte pH 3.5-10 (2% v/v) and a trace of Bromophenol Blue [6].

Sample application
When the rehydration cassette had been thoroughly emptied and opened, the
strips were transferred to the Pharmacia strip tray. After placing IPG strips,
humid electrode wicks, electrodes and sample cups in position, the strips and
cups were covered with low viscosity paraffin oil. Samples were applied at
the cathodic end of the IPG strips in a slow and continuous manner, without
touching the gels [6].

Running conditions
The voltage was linearly increased from 300 to 3500 V during 3 hours,
followed by 3 additional hours at 3500 V, whereupon the voltage was
increased to 5000 V. A total volthourproduct of 100 kvh was used in an
overnight run [6].
IPG gel strips equilibration
After the first dimension run the strips were equilibrated in order to
resolubilize the proteins and to reduce -S-S- bonds. The strips were
equilibrated within the strip tray with 100 ml of a solution containing Tris-
HCl (50 mM) pH 8.4, urea (6 M), glycerol (30% v/v), SDS (2% w/v) and
DTE (2% w/v) for 12 min. -SH groups were subsequently blocked with 100
ml of a solution containing Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol
(30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of
Bromophenol Blue for 5 min [6].

SDS-PAGE as second dimension

In second dimension, a vertical gradient slab gel with the Laemmli-SDS-

discontinuous system was used with some small modifications [13-15].

1. Gels are not polymerized in the presence of SDS. This seems to

prevent the formation of micelles which contain acrylamide monomer,
thus increasing the homogeneity of pore size and reducing the
concentration of unpolymerized monomer in the polyacrylamide. The
SDS used in the gel running buffer is sufficient to maintain the
necessary negative charge on proteins.
2. Piperazine diacrylyl (PDA) is used as crosslinker. We believe this
reduces N-teminal protein blockage, gives better protein resolution,
and reduces diamine silver staining background.
3. Sodium thiosulfate is used as an additive to reduce background in the
silver staining of gels.
4. The combination of the IPG strip and agarose avoids the need for a
stacking gel. In addition, the gels were cast with the Angelique system
from LargeScaleBiology, which it is an efficient and easy to use PC
control equipment that allowed to cast simultaneously 10 to 60 gels.

Gel composition and dimension

Dimension:
160 x 200 x 1.5 mm
Resolving gel:
Acrylamide/PDA (9-16% T / 2.6% C)
Stacking gel:
No stacking
Leading buffer:
 Tris-HCl (0.375 M) pH 8.8
Trailing buffer:
Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3
Additives:
Sodium thiosulfate (5 mM)
Polymerization agents:
TEMED (0.05%)
APS (0.1%)
The gels were poured until 0.7 cm. from the top of the plates and
overlayered with sec-butanol for about two hours. After the removal of the
overlay and its replacement with water the gels were left overnight [15].

IPG gel strips transfer

After the equilibration, the IPG gel strips were cut to size. Six mm were
removed from the anodic end and 14 mm from the cathodic end. The second
dimension gels were overlayered with a solution containing agarose (0.5%
w/v) and Tris-glycine-SDS (25 mM-198 mM-0.1% w/v) pH 8.3 heated at
about 70o C and the IPG gel strips were immediately loaded through it [6].

Running conditions [15]

Current:
40 mA/gel (constant)
Voltage:
The voltage is non-limiting, but usually requires 100 to 400 V.
Temperature:
8-12o C
Time:
5 hours

Protein detection

The application of the 2-D PAGE technology to separate, analyse and

characterize proteins contained in biological samples would not have been
possible without the development of complementary detection methods.
Perhaps today one of the most popular non-radioactive protein detection is
the silver staining which is 100-fold more sensitive than Coomassie Brilliant
Blue staining [6, 14, 16-19]. The different masters shown in SWISS-
2DPAGE were stained with the ammoniacal silver staining as followed:

Silver staining protocol

All steps were performed on an orbital shaker at 36 rpm [6].

1. At the end of the second dimension run, the gels were removed from
the glass plates and washed in deionized water for 5 min.
2. Soaked in ethanol: acetic acid: water (40: 10: 50) for 1 hour.
3. Soaked in ethanol: acetic acid: water (5: 5: 90) for 2 hours or
overnight.
4. Washed in deionized water for 5 min.
5. Soaked in a solution containing glutaraldehyde (1%) and sodium
acetate (0.5 M) for 30 min.
6. Washed 3 times in deionized water for 10 min.
7. In order to obtain homogeneous dark brown staining of proteins, gels
were soaked twice in a 2,7 naphtalene-disulfonic acid solution (0.05%
w/v) for 30 min.
8. Rinsed 4 times in deionized water for 15 min.
9. Gels were stained in a freshly made ammoniacal silver nitrate solution
for 30 minutes. To prepare 750 ml of this solution, 6 g of silver nitrate
were dissolved in 30 ml of deionized water, which was slowly mixed
into a solution containing 160 ml of water, 10 ml of concentrated
ammonia (25%) and 1.5 ml of sodium hydroxide (10 N). A transient
brown precipitate might form. After it cleared, water was added to
give the final volume.
10.After staining, the gels were washed 4 times in deionized water for 4
min.
11. The images were developed in a solution containing citric acid (0.01%
w/v) and formaldehyde (0.1% v/v) for 5 to 10 min.
12.When a slight background stain appeared, development was stopped
with a solution containing Tris (5% w/v) and acetic acid (2% v/v).

Scanning
The Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel) from Molecular
Dynamics and the GS-700 from Bio-Rad have been used as scanning
devices. This scanners were linked to Sparc workstations and Macintosh
computers.
Sample preparation (preparative gels)

Human samples

CEC (Colon epithelial cell): Within a maximum of 30 min, a large right or

left colon tissue sample (5 x 7 cm) was prepared on ice in the operating
room, rinsed with cooled phosphate buffer saline, pH 7.2 to further remove
cell debris and blood and then immersed into an iced PBS solution
containing 3 mM EDTA, 50 µg/ml leupeptine, 0.2 mM PMSF and 0.8 mM
benzamidine. Crypts were scraped away from the basal membrane with a
scalpel. The tissue was then gently pressed through a mesh with a pore size
of 300 µm to separate epithelial cells from stroma and filtered through a
nylon mesh with 200 µm pores. After centrifugation at 350 g at 4o C for 10
min, the membranes were permeabilized in 70 % chilled ethanol and the
cells were shaken overnight at 4o C. After washing with PBS, the cells were
incubated with fluorescein-conjugated anticytokeratin antibodies (CAM 5.2)
for 45 min and then sorted by FACS. The pellet was mixed with 100 µl per
106 cells of a solution containing urea (8 M), CHAPS (4 % w/v), DTE (65
mM), Resolytes 3.5-10 (2 % v/v) and a trace of bromophenol blue. Five
hundred µl of the final diluted colon epithelial cell sample was used for in-
gel sample rehydration.

CSF (cerebrospinal fluid): An aliquot of 3000 µl of human CSF was mixed

with 6000 µl of ice cold acetone and centrifuged at 10000 g at 4o C for 10
minutes. The pellet was mixed with 500 µl of a solution containing urea (8
M), CHAPS (4% w/v), DTE (65 mM), Resolytes 4-8 (2 % v/v) and a trace
of bromophenol blue. The whole final diluted CSF sample was used for in-
gel sample rehydration.

ELC (erythroleukemia cell line): A monolayer culture of human ELC was

grown, rinsed trypsinized and washed as explained in the HEPG2 sample
preparation. A pellet of 107 cells were mixed and solubilized with 500 µl of
a solution containing urea (8 M), CHAPS (4% w/v), DTE (65 mM),
Resolytes 3.5-10 (2 % v/v) and a trace of bromophenol blue. The whole final
diluted ELC sample was used for in-gel sample rehydration.

HEPG2 (hepatoblastoma carcinoma derived cell line): A monolayer culture

of human hepatoblastoma carcimoma cell line (Hep G2) was grown in
Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf
serum (FCS). Cells were rinsed once with DMEM without FCS and were
removed from the flask by incubating them with a solution containing
trypsin (0.5 g) and EDTA (0.2 g). After 3 minutes, DMEM containing FCS
was added into the flask to stop the action of trypsin. The cells were detach
from the surface of the flask by squirting the solution onto the cells. The
suspension was transferred into a tube and the cells were centrifuged at 1000
rpm during 5 minutes. Supernatent was discarded and the cells were washed
with DMEM without FCS. After centrifugation and removal of DMEM, 10
million cells were mixed and solubilized with 0.5 ml of a solution containing
urea (8 M), CHAPS (4% w/v), DTE (65 mM, Resolytes 3.5-10 (2 % v/v)
and a trace of Bromophenol Blue. The whole final diluted HepG2 sample
was used for in-gel sample rehydration.

HEPG2SP (hepatoblastoma carcinoma derived cell line secreted proteins):

Hundred ml of supernatant HEPG2 culture media were concentrated down
to 100 µl in a MicrosepTM Concentrators. The concentrated sample was
mixed with 400 µl of a solution containing urea (8 M), CHAPS (4% w/v),
DTE (65 mM), Resolytes 3.5-10 (2 % v/v) and a trace of bromophenol blue.
The whole final diluted HEPG2SP sample was used for in-gel sample
rehydration. NAME="1001363">HL60 (promyelocytic leukemia cells): A
monolayer culture of a human promyelocytic leukemia cell line was grown
in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf
serum (FCS). Cells were rinsed once with DMEM without FCS and
removed from the flask by incubating them with a solution containing
trypsin (0.5 g/l) and EDTA (0.2 g/l). After 3 minutes, DMEM containing
FCS was added into the flask to stop the action of the trypsin. The cells were
detached from the surface of the flask by squirting the solution onto the
cells. The suspension was transferred into a tube and the cells were
centrifuged at 1000 g during 5 minutes. Supernatant was discarded and the
cells were washed with DMEM without FCS. After centrifugation and
removal of DMEM, 5 x 106 cells were mixed and solubilized with 0.5 ml of
a solution containing urea (8 M), CHAPS (4% w/v), DTE (65 mM),
Resolytes 3.5-10 (2 % v/v) and a trace of Bromophenol Blue. The whole
final diluted HL60 sample was used for in-gel sample rehydration.

KIDNEY: Tissue resections were washed several times in cold rinse buffer
(glutamine-free RPMI 1640 medium containing 5 % fetal calf serum, 0.2
mM phenylmethylsulfonyl fluoridem, 1mM EDTA, and antibiotics:
oxacillin 25 µg/ml, gentamycin 50 µg/ml, penicillin 100 U/ml, streptomycin
100 µg/ml, amphotericin B 0.25 µg/ml, nistatin 50 U/ml) to further remove
cell debris and blood, and were frozen by immersion in liquid nitrogen. They
were then submitted to mechanical dissociation by scraping and gentle
squeezing. The cell suspension was washed several times in cold phosphate
buffer saline, pH 7.2. The cellular pellet was denatured with 100 µl per 106
cells of a solution containing urea (8 M), CHAPS (4 % w/v), DTE (65 mM),
Resolytes 3.5-10 (2 % v/v) and a trace of bromophenol blue. Five hundred
µl containing 5 x 106 kidney cells was used for in-gel sample rehydration.

LIVER Protocole 1: Twenty five frozen slices (20 micrograms x 5 mm x 10

mm) of human liver biopsy were mixed with 500 µl of a solution containing
urea (8 M), CHAPS (4% w/v), DTE (65 mM), Resolytes 3.5-10 (2 % v/v)
and a trace of Bromophenol Blue. The whole final diluted liver sample was
used for in-gel sample rehydration [2 and 3].

LIVER Protocole 2: Ten mg of a human liver frozen biopsy was lyophillized

for 48 h. It was then crushed in a mortar containing liquid nitrogen and
mixed with 500 µl of a solution containing urea (8 M), CHAPS (4% w/v),
DTE (65 mM), Resolytes 3.5-10 (2 % v/v) and a trace of Bromophenol Blue.
The whole final diluted liver sample was used for in-gel sample rehydration.

LYMPHOMA: Twenty frozen slices (20 µm x 5 mm x 10 mm) of a human

lymphoma biopsy were mixed with 500 µl of a solution containing urea (8
M), CHAPS (4% w/v), DTE (65 mM), Resolytes 3.5-10 (2 % v/v) and a
trace of bromophenol blue. The whole final diluted lymphoma sample was
used for in-gel sample rehydration. PLASMA: An aliquot of 250 microliters
of human plasma was mixed with 750 microliters of a solution containing
urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of
Bromophenol Blue. The whole diluted plasma sample (15 mg) was loaded
onto the racket-shaped IPG gel strips [1].

PLATELET: Fresh blood has been centrifuged at 1000 g for 5 minutes. The
supernatant (platelet-rich plasma: RPR) was then centrifuged at 5000 g for
10 minutes. 107 washed platelets were suspended in 5000 µl of lysis buffer
containing Tris-HCl pH 8.0 (10 mM), MgCl2 (1.5 mM), KCl (10 mM), DTE
(0.5 mM) and PMSF (0.5 mM) and incubated on ice for 10 minutes.
Mechanical lysis was achieved with a potter homogenizer and the resulting
lysate was centrifuged at 3000 g for 10 minutes. Then 1/10 volume of a
solution containing Tris-HCl pH 8.0 (0.3 M), KCl (1.4 M) and MgCl2 (30
mM) was added to the supernatant and ultracentrifuged at 54000 g for 2
hours. The supernatant was diluted with 3 volumes of distilled water to
decrease the salt concentration and then concentrated down to 100 µl in a
MicrosepTM Concentrator. The concentrated protein sample was mixed and
solubilized with 400 µl of a solution containing urea (8 M), CHAPS (4%
w/v), DTE (65 mM), Resolytes 3.5-10 (2 % v/v) and a trace of bromophenol
blue. The whole final diluted platelet sample was used for in-gel sample
rehydration [5].

RBC (Red blood cell): Fresh blood was centrifuged at 2500 rpm at 4 C for
10 minutes. Plasma and buffy coat were removed and RBC were washed
three times with the same volume of PBS pH 7.4. An aliquot of 5.4 mg of
RBC was mixed with 500 microliters of a solution containing urea (8 M),
CHAPS (4% w/v), DTE (65 mM), Resolytes 3.5-10 (2 % v/v) and a trace of
Bromophenol Blue.The whole diluted RBC sample was used for in-gel
sample rehydration [4].

U937 (macrophage like cell line): A monolayer culture of human U937 was
grown at a concentration of 0.5 million/ml in RPMI 1640 containing 1%
FCS. The cells were then rinsed, trypsinized and washed as explained in the
HEPG2 sample preparation. After centrifugation and removal of RPMI, 107
cells were mixed and solubilized with 500 µl of a solution containing urea (8
M), CHAPS (4% w/v), DTE (65 mM), Resolytes 3.5-10 (2 % v/v) and a
trace of bromophenol blue. The whole final diluted U937 sample was used
for in-gel sample rehydration.

Mouse samples

BAT (Brown adipose tissue): Eight mg of dried brown adipose tissue was
mixed with 60 µl of a solution containing urea (8 M), CHAPS (4% w/v),
Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of bromophenol
blue. The whole final diluted sample (150 µg) was loaded in a cup at the
cathodic end of the IPG gels.

ISLETS (Pancreatic islet cells): One thousand of small and large pancreatic
islets were mixed with 60 µl of a solution containing urea (8 M), CHAPS
(4% w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of
bromophenol blue. The whole final diluted sample (100 µg) was loaded in a
cup at the cathodic end of the IPG gels.

LIVER: Four mg of dried liver was mixed with 60 µl of a solution

containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM),
SDS (0.05% w/v) and a trace of bromophenol blue. The whole final diluted
sample (100 µg) was loaded in a cup at the cathodic end of the IPG gels.
LIVER NUCLEI (Soluble nuclear proteins and matrix from liver tissue):
Nuclear proteins were solubilized in a buffer containing 5M urea, 2M
thiourea, 2% CHAPS (w/v), 2% sulfobetains (w/v), 65mM DTE, 40mM Tris
(pH 6.8), 0.8% Resolyte 3.5-10 and a trace of bromophenol blue.

MUSCLE (Gastrocnemius muscle): Four mg of dried gastrocnemius muscle

was mixed with 60 µl of a solution containing urea (8 M), CHAPS (4%
w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of
bromophenol blue. The whole final diluted sample (100 µg) was loaded in a
cup at the cathodic end of the IPG gels.

WAT (White adipose tissue): One hundred and sixty mg of dried white
adipose tissue was mixed with 60 µl of a solution containing urea (8 M),
CHAPS (4% w/v), Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a
trace of bromophenol blue. The whole final diluted sample (150 µg) was
loaded in a cup at the cathodic end of the IPG gels.

Other samples

DICTY (Dictyostelium discoideum): A WS380B wild type strain was used

here. Slugs (5 mg dry weight) were resuspended in 500 µl of a solution
containing urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Resolytes 3.5-10
(2 % v/v) and a trace of bromophenol blue. This whole Dicty diluted sample
was used for in-gel sample rehydration.

ECOLI (Escherichia coli): Cells were grown aerobically in glucose minimal

morpholinopropane sulfonate (MOPS), plus thiamine at 37o C. Growth was
stopped in the late exponential phase at an OD of 1 at 600 nm. Five hundred
ml of culture medium was centrifuged for 30 min at 3000 rpm at 4° C and
the pellet was washed 4 times for 10 min at 4000 rpm in 10 ml low salt
washing sample buffer: KCl 3.0 mM, KH2PO4 1.5 mM, NaCl 68 mM,
NaH2PO4 9.0 mM. The pellet was then resuspended in 600 µl of a buffer
containing 10 mM Tris-HCl pH 8.0, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM
DTE, 0.5 mM Pefabloc SC (protease inhibitor), 0.1% SDS, and stored at
-20°C. Fifty µl of the latter was mixed with 450 µl of a solution containing
Urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Resolytes 4-8 (2 % v/v) and
a trace of bromophenol blue. After centrifugation at 10000 g for 5 minutes,
the whole final E. coli diluted sample (5 mg) was used for in-gel sample
rehydration.
YEAST (Saccharomyces cerevisiae): Cells were washed twice with PGSK
(NH2PO4-H2O 0.52 g/l, Na2HPO4-2H2O 8.8 g/l, NaCl 2.83 g/l, KCl 0.372
g/l and glucose 11 g/l), centrifuged at 3000 rpm for 5 min (4oC) and the
supernatant removed. The pellet was resuspended in 1 volume of PGSK and
1 volume of glass beads (425-600 µm diameter) and shaked for 10 min at
4oC in a bead beater. The extracts were centrifuged at 3000 rpm for 10 min
at 4oC, the supernatant retained and the pellet subjected to procedures (c)
and (d) a second time. The supernatants were pooled and 1-5 mg (measured
by modified Lowry) of yeast proteins was mixed with 500 µl of a solution
containing urea (8 M), CHAPS (4 % w/v), DTE (65 mM), Resolytes 4-8 (2
% v/v) and a trace of bromophenol blue. The whole yeast diluted sample
was used for in-gel sample rehydration.

Immobilized pH gradient (IPG) as first dimension

Racket-shaped IPG

A new sigmoidal immobilized pH gradient (IPG) was used as the first

dimension. It offered high resolution and great reproducibility and allowed
high protein loads. Based on our specifications, the non-linear pH gradient
strips were prepared by Pharmacia LKB Biotechnology AB and are
commercially available. The strips were 3 mm wide and 180 mm long. In
addition, the geometry of the immobilized pH gradient strips has been
changed to allow the use of large sample application cups that can
accommodate greater sample volumes (see sample preparation). The use of
narrow range IPG with a large sample loading volume allowed an efficient
resolubilisation of polypeptides after the first dimension. As a result, the
vertical streakings caused by too high a protein concentration were
eliminated in the second dimension [6-12].

Narrow IPG gel strips preparation

A variety of recipes were used giving IPG's with a width of 0.4 to 1 pH
units. The strips were cut in a form to allow sample application in sample
cups of 16 x 11 x 6 mm [8].
IPG gel strips rehydration
Hydration was performed overnight in the Pharmacia reswelling cassette
with 25 ml of a solution containing urea (8 M), CHAPS (2% w/v), DTE (10
mM), Resolyte pH 3.5-10 (2% v/v) and a trace of Bromophenol Blue [8].

Sample application
When the rehydration cassette had been thoroughly emptied and opened, the
strips were transferred to the Pharmacia strip tray. After placing IPG strips,
humid electrode wicks, electrodes and sample cups in position, the strips and
cups were covered with low viscosity paraffin oil. Samples were applied at
the cathodic end of the IPG strips in a slow and continuous manner, without
touching the gels [8].

Running conditions
The voltage was linearly increased from 300 to 3500 V during 3 hours,
followed by 3 additional hours at 3500 V, whereupon the voltage was
increased to 5000 V. A total volthourproduct of 400 kvh was used in a four
days run [8].

IPG gel strips equilibration

After the first dimension run the strips were equilibrated in order to
resolubilise the proteins and to reduce -S-S- bonds. The strips were
equilibrated within the strip tray with 100 ml of a solution containing Tris-
HCl (50 mM) pH 8.4, urea (6 M), glycerol (30% v/v), SDS (2% w/v) and
DTE (2% w/v) for 12 min. -SH groups were subsequently blocked with 100
ml of a solution containing Tris-HCl (50 mM) pH 6.8, urea (6 M), glycerol
(30% v/v), SDS (2% w/v), iodoacetamide (2.5% w/v) and a trace of
Bromophenol Blue for 5 min[8].

In-gel sample rehydration

Two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) is one of

the most powerful technique for the study of protein expression and their
post-translational modifications. However, the separation of low copy
number proteins in amounts sufficient for post-separation analysis continues
to present a challenge for 2-D techniques. Improvements in shortening the
focusing time, increasing the loading capacity and enhancing resolution is
still needed. We developed a simple methodology for sample application
suitable for commercially available or home-made IPG strips. To achieve
this, we have adapted the concepts of "volume-controlled rehydration" and
"in-gel sample rehydration", but modified the approaches to exploit the
reproducibility offered by commercially available IPG strips.

In our method, the entire IPG gel is used for sample application, with the
protein entering the gel during its rehydration. A methacrylate reswelling
chamber was built to accomodate 10 IPG strips in separate grooves. A
schematic diagram of the chamber is shown here.

Each groove was 10 mm deep, 4.0 mm wide and 205 mm long. Four
levelling feet and a spirit level were included in the chamber. This chamber
sould be easily constructed in workshops.

IPG gel strips rehydration and sample application

Hundred µg to 15 mg of proteins were solubilized with 500 µl of a solution
containing 8 M urea, 4% CHAPS, 65 mM DTE, 0.8% resolytes 4-8 and a
trace of bromophenol blue. To achieve reswelling and simultaneous loading
of the sample, the entire samples were pipetted into the grooves, narrow (1
pH unit) or wide range IPG strips (3.5-10 NL, 18 cm from Pharmacia
Biotech. or home-made) were positioned such that the gel of the strip was in
contact with the sample (up side down), and the gel and the sample were
covered with 3 ml low viscosity paraffin oil to avoid evaporation. Strips
were then left at room temperature for six hours or overnight. The
rehydrated IPG gels carrying the protein sample were removed from the
grooves with tweezers, rinsed with water and positioned on the Pharmacia
strip tray as described by the manufacturer.
Running conditions
The voltage was linearly increased from 300 to 3500 V during 3 hours,
followed by 3 additional hours at 3500 V, whereupon the voltage was
increased to 5000 V. Focusing was carried out for a total of 100 kVh.

After running, strips can be frozen (-20oC) for several weeks (remove oil), or
used immediately for the second dimension.

IPG gel strips equilibration

After the first dimension run the strips were equilibrated in order to
resolubilise the proteins reduce -S-S- bonds. They were equilibrated for 12
min in the reswelling chamber using 3 ml per groove of buffer 1 (50 mM
Tris-HCl pH 6.8, 6 M urea, 30% v/v glycerol, 2% w/v SDS and 2% w/v
DTE), and then -SH groups were subsequently blocked for 5 min using
buffer 2 (50 mM Tris-HCl pH 6.8, 6 M urea, 30% v/v glycerol, 2% w/v
SDS, 2.5% w/v iodoacetamide and a trace of bromophenol blue). Buffers are
easily removed by aspiration.

Protein electroblotting

The blotting of proteins separated by two-dimensional polyacrylamide gel

electrophoresis onto polyvinylidene difluoride (PVDF) membranes [3 and
20] has enabled the identification and characterization of proteins from
complex biological samples. Transfer of the proteins can be carried out
using several methods such as vacuum, capillary or electric field.
Electroblotting is by far the most wide-spread technique which utilizes either
vertical buffer tanks or semi-dry blotting. Both techniques can use either the
Towbin or 3-[cyclohexamino]-1-propanesulfonic acid (CAPS) transfer
buffers, depending on the need for minimal glycine contamination in post-
transfer protein characterization. These two buffer systems are described
here:

Gloves must be worn and all filter papers should be washed three times for 3
min in water and three times in transfer buffer. These two steps are
important in order to avoid any protein or amino acid contamination.
Towbin buffer system

1. After second-dimensional electrophoresis, soak the gels in deionized

water for 3 min.
2. Equilibrate the gels in a solution containing Tris (13 mM), glycine
(100 mM) and methanol (10% v/v) for 30 min. At the same time, wet
PVDF membranes in methanol for 1 min and equilibrate them in a
solution containing Tris (13 mM), glycine (100 mM) and methanol
(10% v/v) also for 30 min.
3. Carry out electroblotting either in:
1. a transfer tank with a solution containing Tris (13 mM), glycine
(100 mM) and methanol (10% v/v) at 90 V constant voltage for
3 hours at 15oC. Assemble the blotting sandwich as described in
chapter 5 of this book.
2. or a semi-dry apparatus with a solution containing Tris (13
mM), glycine (100 mM) and methanol (20% v/v anodic side;
5% v/v cathodic side) at 1 mA/cm2 constant current for 3 hours
at 15oC or as described by the manufacturer.

CAPS buffer system

1. After second-dimensional electrophoresis, soak the gels in deionized

water for 3 min.
2. Equilibrate the gels in a solution containing 10 mM CAPS pH 11 for
30 min. At the same time, wet PVDF membranes in methanol for 1
min and equilibrate them in a solution containing 10 mM CAPS pH
11 and methanol (10% v/v) also for 30 min.
3. Carry out electroblotting in either:
1. a transfer tank with a solution containing 10 mM CAPS pH 11
and methanol (10% v/v) at 90 V constant voltage for 3 hours at
15oC. Assemble the bloting sandwich as described in chapter 5
of this book.
2. or a semi-dry apparatus with a solution containing 10 mM
CAPS pH 11 and methanol (20% v/v anodic side; 5% v/v
cathodic side) at 1 mA/cm2 constant current for 3 hours at 15oC
or as described by the manufacturer.
Protein detection onto PVDF membranes

2-D PAGE and electroblotting onto PVDF membranes have become widely
used techniques for the characterization of proteins. Recent improvements
have allowed a higher protein load, a better transfer yield and a higher
sensitivity in automated protein microsequencing. However, the application
of these techniques to proteins would not have been possible without the
development of complementary detection methods. Amido Black,
Coomassie Brilliant Blue R-250, colloidal gold and Ponceau S are
commonly utilized to visualize proteins on PVDF membranes and are
compatible with the ensuing Edman degradation chemistry [3].

Amido Black
After electrotransfer, the PVDF membranes were stained in a solution
containing Amido Black (0.5% w/v), isopropanol (25% v/v) and acetic acid
(10% v/v) for 2 min. Destaining was done by several soakings in deionized
water [3].

Coomassie Brilliant Blue R-250

After electrotransfer, the PVDF membranes were stained in a solution
containing Coomassie Brilliant Blue R-250 (0.1% w/v) and methanol (50%
v/v) for 15 min. Destaining was done in a solution containing methanol
(40% v/v) and acetic acid (10% v/v) [3].

Colloidal Gold (Progold)

After electrotransfer, the PVDF membranes were incubated in PBS-Tween
0.5% for 30 minutes, washed 3 x 5 min. in PBS-Tween 0.5% and 1 min. in
deionized water. They were stained in 100 ml of Problot solution overnight.

Ponceau S
After electrotransfer, the PVDF membranes were stained in a solution
containing Ponceau S (0.2% w/v) and TCA (3% v/v). Destaining was done
by several soakings in deionized water [3].

Drying
The PVDF stained membranes were either air dried or dried on a 3 mm thick
plate onto an heating plate at 37o C for 10 min [3].
Scanning
The Laser Densitometer (4000 x 5000 pixels; 12 bits/pixel) from Molecular
Dynamics and the GS-700 from Bio-Rad have been used as scanning device.
This scanners were linked to Sparc workstations and Macintosh computers.

Sequencing

Amino sequence analysis using Edman degradation is one of the most

important techniques for the investigation of proteins at the molecular level.
Amino acid derivatives are sequentially cleaved one at a time from the
protein. Proteins with a chemically inaccessible alpha-amino group cannot
be sequenced directly by this procedure and are termed N-terminally
blocked. The best way to overcome the blocked proteins is to generate
individual fragments by chemical or proteolytic cleavage [21-26] or to
analyze the amino acid composition.

N-terminal sequencing
The Amido Black stained proteins were excised with a razor blade and N-
terminal sequence determination were performed using either ABI model
473A or 477A microsequencers from Applied Biosystems equipped with
Problott cartridges.

Internal sequencing
The spots of interest were excised and soaked two hours in a solution
containing acetic acid (100 mM), methanol (10% v/v) and PVP-40 (1% v/v)
at 37 C. After three washes in deionized water, the PVDF spots were cut into
small pieces (~1 square millimeter) and incubated in 25 microliters of a
solution containing sodium phosphate (100 mM) pH 8.0 and lysyl
endopeptidase (1 microgram). Following overnight digestion at room
temperature, guanidine-HCl (28 mg) and DTT (100 micrograms) were
added. After reduction for 2 hours at 37 C, the mixture was incubated for 30
min, at room temperature, with 300 micrograms of iodoacetamide. The
digestion solution was removed and guarded. PVDF pieces were then
extracted overnight with 25 microliters of a solution containing isopropanol
(70% v/v) and trifluoroacetic acid (5% w/v). This elution solution was
removed and the PVDF was washed twice with 60 microliters of TFA (0.1%
w/v). The digestion and elution solutions were pooled together with two
final washes and this mixture was separated by two-dimensional reverse
phase HPLC and sequence determination performed.
Routinely, ten to twelve Edman degradation cycles were performed for each
spots. A search in the Swiss-Prot [Ref. 27] database was made to detect
identity to known protein sequences (see TagIdent tool for details).

Amino acid composition

There has been a recent revival of interest in the use of AA composition for
the identification of proteins from 2-D gels. This technique uses the
idiosyncratic AA composition profile of a protein in order to identify it by
comparison with theoretical AA compositions of proteins in databases. For
identification of proteins from 2-D gels, we match the AA composition in
conjunction with estimated protein pI and Mw. Protein identification by
compositional analysis is best used when there is sufficient sample available
for micropreparative 2-D PAGE, as a minimum of 250 ng of protein per spot
of interest is required. As the approach is rapid, inexpensive, and produces
easily interpreted data, it is suited for the screening of large numbers of
proteins from 2-D reference maps. We can analyze 20 PVDF-bound proteins
per day on a single AA analysis station. Rapid methods for the AA analysis
of PVDF-bound proteins are presented below. These methods have been
optimized for use with samples prepared by micropreparative 2-D and
blotted to PVDF in a glycine-free buffer. To control for variation in AA
analysis results, we always analyze samples in batches. Each batch
comprises a calibration protein (PVDF-bound bovine serum albumin) and 12
samples. The batch is hydrolyzed together, AAs are extracted using common
solutions, and the AA analysis of each batch is carried out sequentially on
the analysis instrument. After AA analysis, the analysis quality of the
calibration protein is checked as a benchmark, and its analysis data is used to
adjust that from unknown spots during protein identification by database
searching.

Hydrolysis of PVDF-bound proteins

Vapor-phase protein hydrolysis requires that you have a hydrolysis vessel,
and vessel holder. This design is recommended as other vessels (even some
which are commercially available) are not able to withstand repeated heating
to 155oC in the presence of HCl vapor. Access to a vacuum source and fume
hood are also required. At all times, contamination of samples with dust,
skin, hair and breath must be avoided. It is advisable to wear powder-free
latex gloves and work in a clean environment.
Vapor-phase hydrolysis of protein spots from 2-D gels for amino acid
analysis

1. Cut spots of interest from PVDF membranes with a scalpel or cold

punch, on a minimum of PVDF.
2. Place each spot into a 700 µl clear glass autosampler vial. If possible,
use vials that can later be used directly on your AA analysis
autosampler. If spots are very feint and/or small, multiples of identical
spots from different gels can be placed into a single vial. Use a
diamond pen or glass engraving tool to label vials.
3. Place 400 µl of 5.7 N HCl (BDH Aristar Grade) and a crystal of AR-
grade phenol (approximately 3 mm long by 0.5 mm wide) into the
bottom of the hydrolysis vessel. Note that these reagents do not go
into the autosampler vials containing the samples.
4. Using a pair of stainless steel tweezers, place up to 13 autosampler
vials into the hydrolysis vessel so they are upright. Include one vial
containing a calibration protein.
5. Assemble the hydrolysis vessel tightly. Evacuate the vessel for 10 sec
(the acid should boil), and then flush with argon. Repeat the
evacuation / flush steps. Finally evacuate, close the tap on the vessel,
and place the vessel into the preheated vessel holder in the 155o C
oven for 1 h.
6. After heating, remove the vessel from the oven and transfer it to a
fume hood. Release the acid vapor from the vessel by opening the tap,
dismantle the vessel, and remove the autosampler vials with tweezers.
These steps should be done immediately to prevent condensation of
acid forming on the samples. CAUTION: Vessels must be opened in a
fume hood, as hot HCl vapor is very dangerous! Eye and hand
protection must be worn!
7. Dry the autosampler vials containing the PVDF membranes under
vacuum for 10 min (Savant Speedvac) to remove residual HCl vapor.

Post-hydrolysis extraction of amino acids from PVDF

After hydrolysis of PVDF-bound proteins, AAs are extracted from the
membranes in preparation for AA analysis. To minimize sample
manipulation, samples are kept in the same vial for the entire extraction
procedure. A sonicating water bath is required. Note that step (e) of this
protocol resuspends the extracted AAs in 250 mM sodium borate buffer pH
8.8 in preparation for AA analysis using 9-fluorenylmethyl chloroformate
(Fmoc). This buffer may, however, not be suitable if AA analysis is to be
done using other derivatisation chemistries.

Post-hydrolysis extraction of amino acids from PVDF

1. Add 180 µl of fresh extraction solution (60% v/v acetonitrile in 0.01%

v/v trifluoroacetic acid) to each autosampler vial containing a PVDF
spot. Make sure the PVDF is submerged in the solution.
2. Cover each vial with parafilm, position in a polystyrene floater, and
place the floater into a sonicating water bath (21o C) for 10 min.
3. Discard the parafilm covers of the vials. Remove the PVDF
membrane from each autosampler vial with a hypodermic needle and
discard. Keep the hydrolysate in the autosampler vial. Vial to vial
sample carryover should be avoided by either using only one needle
per vial or rinsing the needle between vials in fresh extraction
solution.
4. Place the autosampler vials containing protein hydrolysates in a
vacuum dryer (Savant Speedvac) and evaporate to dryness.
5. Add 10 to 20 µl 250 mM sodium borate buffer pH 8.8 to each
autosampler vial, mixing carefully to ensure all AAs are dissolved.
The samples are now ready for AA analysis.

Derivatisation and chromatography

The AA analysis of protein hydrolysates is achieved using a modified Fmoc
precolumn derivatisation method which is carried out at room temperature,
produces monosubstitued forms of His and Tyr, and does not require the
removal of excess Fmoc before chromatography. Derivatisation of AAs
should be carried out in the same glass vial that was used for hydrolysis and
extraction. We derivatise AA standards (Sigma # AA-S-18) to check
derivatisation and chromatography efficiency and to allow quantitation of
samples. Note that the Fmoc-amino acid derivatives are stable for 24 h,
allowing many samples to be prepared in advance and loaded onto an
autosampler for injection. Alternatively, the derivatisation can be done by
any autosampler which has minimal vial to vial sample carryover and can
accurately manipulate 10 µl volumes. We use a GBC Aminomate Amino
Acid Analyzer (GBC Scientific Instruments, Dandenong, Vic., Australia) for
this purpose.
Derivatisation of amino acids with 9-fluorenylmethyl chloroformate

1. Ensure that all reagents and samples are at room temperature.

2. Starting with 10 µl of hydrolysate, add 10 µl of Fmoc reagent and mix
thoroughly by pipetting up and down. Wait for 90 sec.
3. Add 10 µl of cleavage reagent and mix thoroughly by pipetting. Wait
for 3 min 30 sec.
4. Add 10 µl of quenching reagent and mix thoroughly by pipetting.
5. The mixture can now be injected directly into the HPLC system, or
sealed and stored for up to 24 h.

Amino acid composition was then matched, in conjunction with the

estimated pI and molecular weight of the protein with the corespondent
protein in Swiss-Prot database. The matching algorithm calculates the least
squared distance between the theoretical and measured amino percentage for
each protein (see AACompIdent tool for details). A score was obtained and a
typical pattern was defined for assignments.

Sequence Tag

Automated Edman degradation, which produces N-terminal protein

sequence, is a common identification method for PVDF-bound proteins.
Sequencing is usually done for 15 cycles, and identity is established by
matching the sequence obtained against those in protein databases. Internal
protein sequencing is also possible, but is a more labor-intensive procedure.

In our early work with 2-D PAGE reference maps we almost exclusively
used N-terminal sequence analysis for protein identification. However, with
a throughput of one protein per sequencer per day, this approach was too
slow for the identification of large numbers of protein samples. To address
this problem but continue to use the specificity of protein sequence data, we
now use a combination of Edman degradation and Amino acid analysis
techniques for rapid protein identification. PVDF-bound proteins are
sequenced for only three or four cycles at the amino terminus, to create a N-
terminal sequence tag. We use fast but low repetitive yield sequencing
programs as only a few residues of sequence are required. After sequencing,
the same PVDF-bound protein sample which was sequenced is used for AA
analysis. Protein identification is then achieved by matching AA
compositional data, estimated protein pI and Mw, and the protein "sequence
tag" against the Swiss-Prot database (see AACompIdent tool for details).
This approach provides a powerful and unambiguous identification method
which, as compared to extensive N-terminal protein sequencing, increases
sample throughput five to ten fold and greatly reduces reagent costs.

Peptide mass fingerprinting and amino acid composition identification

Mass spectrometry is a rapidly growing field of protein analysis, which is

proving useful in the identification of proteins separated by 2-D gel
electrophoresis. The most common mass spectrometry protein identification
technique is called peptide mass fingerprinting. This involves the generation
of peptides from proteins using residue-specific enzymes, the determination
of peptide masses by spectrometric techniques, and the matching of these
masses against theoretical peptide libraries generated from protein sequence
databases to create a list of likely protein identifications.

We do not currently use peptide mass fingerprinting alone for protein

identification, but often use an identification approach which compares lists
of best-matching proteins generated by AA composition identification and
peptide mass fingerprinting techniques (see MultiIdent tool for details). For
this approach, one sample is subjected to the amino acid analysis procedure
to generate a list of best matching proteins, and a duplicate sample is
subjected to peptide mass fingerprinting to generate an independent list of
best matching proteins. Identification is achieved when lists of best-
matching proteins are then compared for identical database. This technique
has two main applications. Firstly, in situations where proteins are blocked
at the N-terminus and cannot be used for "sequence tagging", and secondly
where proteins are being identified over large or small phylogenetic
distances across species boundaries. As peptide mass fingerprinting has a
sample throughput similar to AA analysis, this combined identification
approach is suitable for rapid protein identification.

Immunoblotting

Immunodetection is a powerful and sensitive technique, which relies on the

specificity of antibodies to identify single protein spots from 2-D PAGE.
The technique we currently use for immunodetection protein identification is
enhanced chemiluminescence (ECL). With this method, PVDF membranes
are first stained to visualize proteins, following which the immunodetection
is undertaken. This allows matching of proteins detected with ECL against
those detected with the non-specific protein stain through computer
comparison of both images. The mechanical strength of PVDF is also
exploited as the same 2-D gel can be used many times for different
antibodies. Immunoblotting is good to use where only small quantities of
sample are available as it can detect as little as picogram amounts of protein,
depending on the specificity of the antibodies. However, it is a slow
technique, as it is only possible to identify a few proteins per gel per day. It
also requires the prior existence of monoclonal or polyclonal antibodies,
which may be expensive to make or obtain commercially. In the below
protocol, we often probe with 8 or 9 antibodies at once to increase protein
identification rates. However, in such cases we first check that there is
sufficient pI and Mw difference in the proteins of interest to avoid
identification ambiguities.

Enhanced chemiluminescence (ECL) immunodetection procedure

We carry out the whole procedure in a rotating oven at room temperature.
The use of a nucleic acid glass hybridizer tube minimizes the volumes and
costs.

1. Block the membrane in 10 ml of a solution containing PBS (pH 7.2)

and nonfat dry milk (5% w/v) for 30 min.
2. Incubate the membrane in 10 ml of a solution containing PBS-Tween
20 (0.5% v/v), nonfat dry milk (5% w/v) and the primary
antibody/antibodies (1:100 or greater, depending on Ab) for 2 h.
3. Perform three quick rinses with 10 ml of PBS-Tween 20 (0.5% v/v)
and then wash the membrane for 3 x 10 min with 10 ml of PBS-
Tween 20 (0.5% v/v).
4. Incubate the membrane in 10 ml of a solution containing PBS-Tween
20 (0.5% v/v), nonfat dry milk (5% w/v) and the secondary
peroxidase-conjugated antibody (1:1000; for example, if the primary
antibody was mouse anti-human, then use goat anti-mouse IgG) for 1
h.
5. Perform three quick rinses with 10 ml of PBS-Tween 20 (0.5% v/v)
and then wash the membrane for 5 x 10 min with 10 ml of PBS-
Tween 20 (0.5% v/v).
6. After the last wash, transfer the membrane to a clean glass plate and
cover the membrane with 10 ml of developing solution (for example
ECL from Amersham International or Borhinger Manneihm) for 2
min.
7. Drain the excess developing solution and wrap the membrane in
SaranWrap. Fix it in an x-ray film cassette with the proteins facing up.
 8. Go to the dark room and expose an x-ray film for few seconds or up to
several minutes.

-D Polyacrylamide Gel Electrophoresis

This method was successful in our lab using prostate tissue and for our
specific objectives. Investigators must be aware that they will need to
tailor the following protocol for their own research objectives and tissue
under study.

Solutions

TIP: Use electrophoresis grade reagents to prepare the following solutions:

A: 50 ml IEF Lysis Buffer

1. Add 21 g urea to 35 ml HPLC-grade H2O to a 50 ml Falcon tube (final

concentration 7 M).
2. Vortex vigorously for several minutes.
3. Add:
7.6 g Thiourea
2g Chaps
0.5 g Mega 10
0.5 g OBG
250 µl Triton X-100
0.25 g Tris
0.4 g DTT
Pharmalytes or IPG buffer pH 3-10
500 µl
(Amersham)
500 µl - mercaptoethanol
4. Add 10 µl tributylphosphine 2 mM, under the hood (2 mM final
conc.)
5. Add Bromophenol Blue as indicator.
6. Check volume is 50 ml.
7. Vortex until all is dissolved (or attach tube to a rotator).
8. Aliquot 1 ml into microfuge tubes.
9. Store at -20°C.

B: 10X Electrophoresis Running Buffer (10 L) 0.25 M Tris, 1.92 M

glycine, 1M SDS
 1. Add 300 g Tris-base, 1441 g glycine, and 10 g SDS to ~7 L HPLC-
grade H2O.
2. Mix gently until dissolved.
3. Bring volume to 10 liters.

C: 30% Acrylamide Stock (1 liter)

1. Add 292 g acrylamide and 8 g piperazine diacrylamide (PDA)

to 700 ml HPLC-grade H2O, under the hood.
2. Stir to dissolve.
3. Bring volume to 1 L.
4. Filter through 0.45 µm pore size filter.
5. Store at 4°C in the dark.

D: Separating Acrylamide Gel

Below are the solution volumes required to prepare one 9-18% gradient gel.
Prepare sufficient volume for the number of gels to be run.

Solution Volum 9% gel 18% gel

e Units
1.5 M Tris-HCl, ml
11.5 11.5
pH 8.8
20% SDS ml 0.23 0.23
30% Acrylamide ml 14 28
TEMED µl 11.7 11.7
10% APS µl 117 117
HPLC-grade H2O ml 20 6
Total ml 45.8 45.8

E: 50 ml Equilibration Buffer I

1. Mix together 18 g urea and 10 ml of 0.5 M Tris-HCl, pH 6.9.

2. Vortex vigorously.
3. Add 10 ml of 20% SDS and 200 mg DTT.
4. Invert gently several times.
5. Add 15 ml glycerol.
 6. Vortex vigorously (or attach tube to a rotator for 10-15 min).
7. Add Bromophenol Blue as indicator.

F: 50 ml Equilibration Buffer II

1. Same as Equilibration Buffer I EXCEPT add 5.0 g Iodoacetamide

INSTEAD of DTT.

G: Transfer Buffer

1. For 1 L:
25 mM Tris-HCl (3 g)
190 mM glycine (14.44 g)
20% methanol (200 ml)

Day One: Sample Preparation

A. Tissue Processing (See Limitations of 2D-PAGE Electrophoresis for

number of cells needed)

• To lyse a 5-8 µm tissue section obtained from a paraffin-embedded

block:

1. Place the tissue section in a 1.5 ml Eppendorf tube.

2. Add xylenes to cover the tissue.
3. Vortex vigorously for ~15 sec.
4. Incubate at RT for 5 min.
5. Vortex again.
6. Spin down for 3 min to pellet the tissue.
7. Remove xylenes.
8. Add 1 ml xylenes.
9. Vortex 5-10 sec.
10.Spin down.
11.Remove xylenes.
12.Speed vacuum the sample for a few minutes to evaporate the
remaining xylene.
13.Add 400 µl IEF buffer.
14.Vortex vigorously for 1 min.
15.Incubate for 5 min at RT.
16.Vortex vigorously for 1 min.
17.Spin sample down at 14,000 g for 5-10 min at room temp.
• To lyse a 5-8 µm tissue section obtained from a polyester-embedded
block:

Follow 1-15 above for a paraffin-embedded block section, EXCEPT

use 100% EtOH instead of xylenes.

• To a 5-8 µm tissue section obtained from a frozen block OR a

microdissected tissue sample:

1. Place the tissue section or microdissected tissue sample in a 1.5 ml

Eppendorf tube.
2. Add 400 µl IEF buffer.
3. Vortex vigorously for 1 min.
4. Incubate for 5 min. at RT.
5. Vortex vigorously for 1 min.
6. Spin sample down at 14,000 g for 5-10 min at room temp.

• To lyse a paraffin-embedded section on a slide:

1. Deparaffinize the tissue by immersing the slide into xylenes, twice for
5 min each.
2. Allow the tissue section to dry.
3. Add 200 µl of the IEF buffer to the tissue and pipet up and down
several times.
4. Remove the buffer into a microfuge tube.
5. Scrape the tissue with a razor blade and transfer into the same
microfuge tube.
6. Add 200 µl to the lysates for a total of 400 µl.
7. Vortex vigorously for 1 min.
8. Incubate for 5 min at RT.
9. Vortex vigorously for 1 min.
10.Spin sample down at 14,000 g for 5-10 min at room temp.

• To lyse a polyester-embedded section on a slide:

1. Remove the polyester by immersing the slide into ethanol, twice for 5
min each.
2. Repeat steps 2-10, directly above, as for a paraffin-embedded section
on a slide.

• To lyse a frozen section on a slide:

 1. Thaw the slide to room temperature.
2. Immerse the tissue section into xylenes for 1 min.
3. Repeat steps 2-10, directly above, as for a paraffin-embedded
section on a slide.

B. Reswelling

1. Remove Immobiline Drystrips (Amersham Pharmacia Biotech) from

-20°C and allow to equilibrate at RT.

TIP: The pH range of the strip used should be the same as the pH
range of Pharmalytes or IPG buffer used in the IEF lysis buffer.

2. Notch the basic end of the strip to mark each sample.

3. Load the first sample into Reswelling trays (Immobiline DryStrip
Reswelling Tray/Pharmacia #18-1004-31.)
4. Place DryStrips gel-side down into each slot.
5. Remove air bubbles by pressing down with a pipette tip.
6. Overlay completely with DryStrip Cover fluid (Amersham Pharmacia,
#17-1335-01).
7. Repeat for every sample including the MW standard (2-D SDS-PAGE
standards, pH range 4.5-8.5, MW 17,500-76,000, Bio-Rad, # 161-
0320).
8. If samples are concentrated in one region of the strip, redistribute by
pipetting.
9. Cover the tray with the lid.
10.Incubate overnight at RT to allow the strips absorb the samples.

Day Two: 1st Dimension

1. Clean the electrophoresis chamber (Pharmacia LKB Multiphor II) and

the Immobiline strip tray and wipe out with paper towels and
Kimwipes to remove mineral oil.
2. Place the tray on top of 50 ml DryStrip Cover fluid.
3. Remove strips.
4. Place on Whatman paper gel-side up.

TIP: Placing the strips gel-side down might result in protein loss and
gel damage.
 5. Leave for ~1 min.
6. Place into the electrophoresis chamber gel-side up.
7. Arrange the strips so that their edges are in one line.

TIP: Time is important to prevent crystallization.

8. Wet the pre-made IEF electrode strips (Amersham Pharmacia, #18-

1004-40) with HPLC-grade H2O.
9. Dry slightly between two pieces of Whatman paper.
10.Place 2 buffer strips on both edges of the strips and perpendicular to
them, covering the top of the bromophenol blue on each side.
11.Make sure that the square end of each strip is at the cathode (Black/-)
end and the pointed end is at the anode (Red/+) end and also that the
anode and cathode electrode ridges are in the correct orientation.
12.Overlay liberally with DryStrip Cover fluid between the immobilon
strips and outside the electrodes.
13.Electrophorese for 36-48 hrs, using the following sequence of
settings:
Voltag Wattag
Amps Time
e e
100
1 500 V 33 W 0.05 hrs
mA
110
2 500 V 70 W 1 hr
mA
141
3 3500 V 32 W 5 hrs
mA
Until
4 3500 V 70 mA 38 W
stopped
14.The bromophenol blue should be seen migrating towards the anode
within at least 1 hr. By next day, the strips should be colorless.
15.If by the next day the bromophenol blue has not disappeared, running
can be paused and the electrode strips can be replaced from whichever
side. Continue running until the dye has disappeared.

Day Three: 2nd Dimension

A: Prepare Apparatus
 1. Wash and scrub plates very well in soap and hot water.
2. Rinse in diH2O.
3. Leave the plates to air dry or wipe with methanol-soaked Kimwipes.
4. Order plates in Protean-II Multi-Gel casting Chamber (Bio-Rad,
#165-2025) as follows:
Bottom of
chamber
Small plate, 20 cm
Spacers, 20 cm x 1
mm
Eared plates, 20
cm
Spacers, 20 cm x 1
mm
Large plate, 20 cm
Meylar sheet
Repeat as needed.
5. Fill the chamber with acrylic blocks.
6. Tighten the screws.
7. Tape the edges of the chamber to prevent leakage.

B: Prepare Gradient Acrylamide Gel (9-18%)

1. Add 9% gel solution to the center compartment of the distributor

(mixing chamber) and 18% gel to the peripheral compartment
(reservoir chamber).
2. Start the magnetic stirrer in the mixing chamber.
3. Remove air bubbles from the tubing by opening the valve slowly.
4. Allow the tubing to fill with gel solution, then close the valve.
5. Turn on the stirrer.

TIP: The stir bar should be between the openings between the mixing
and reservoir compartments, but not on top of them.

6. Open the valve between the mixing and the reservoir chambers
(upward).
7. Make sure the 18% solution is flowing into the mixing compartment.
8. Open the valve to start the flow of the acrylamide solution into the
Protean chamber.
 9. Allow reasonable flow. Fast flow results in loss of a gradient, whereas
a slow flow results in polymerization of the solution in the tubing. In
addition, the rate of flow changes with time due to the change of
pressure. However, the chamber should be filled in at least 10
minutes.
10.Stop when the gel has reached 0.5 cm from the top of the glass plates.
11. Overlay the gels carefully with HPLC-grade H2O using a syringe.
12.Cover with Saran Wrap.
13.Allow to polymerize overnight.

Day Four: 2nd Dimension (cont.)

1. Remove strips.
2. Place on Whatman paper, gel-side up, for 1 min.
3. Equilibrate in Equilibration Buffer I for 10-15 min.
4. Set-up gels in electrophoresis chamber (Protean II Multi-cell, Bio-
Rad).
5. Make sure there is no leakage.
6. Equilibrate strips in Equilibration Buffer II.
7. Remove strips.
8. Place on Whatman paper one by one, gel-side up.
9. Identify notches.
10. Cut ~one inch from both sides.
11.Place gels with basic side closer to the anode (Red/+) and the acidic
side closer to the cathode (Black/-) to be consistent.
12.Run gels at 40 mA/gel for 15 min or until dye front is about an inch
into the gel.
13.Turn down voltage and run at 65-70 V constant overnight. (If in a
hurry, set power supply at 360 mA and 500 V and run for 8 hrs).

Day Five: Transfer and Sequencing of Proteins

A: Transfer onto PVDF or Nitrocellulose Membranes

1. Soak gel in transfer buffer for 5-10 min.

2. Soak pre-cut transfer membrane and Whatman paper in transfer
buffer.
3. Wet the transfer apparatus with transfer buffer.
4. Place 3-4 Whatman papers on bottom of the transfer apparatus.
5. Place membrane on top and remove air bubbles.
 6. Place gel on top of the membrane and remove air bubbles.
7. Place 3-4 Whatman papers on top of the gel and remove any air
bubbles.
8. Transfer for 3 hrs at no more than 25V (1-1.5 mA per cm 2 of the
gel/membrane). Transfer for 5 hrs if more than 5-6 gels.

B: Gel Staining

See Staining Protocols

C: Excision of Protein Spots (for sequencing by MS)

TIP: Work under the hood to prevent the gel from being exposed to too
much air, thus avoiding contamination with keratins

1. Xerox the gel and assign the spot(s) to be sequenced.

2. Cut out the protein spot for sequencing with a pipette tip.
3. Remove to a microfuge tube.
4. Chop up the gel piece with pipette tip.
5. Add a solution of 50% methanol/10% acetic acid to the gel
pieces.
6. Incubate for 30 min.
7. Spin down.
8. Remove the supernatant.
9. The sample is ready for Mass Spectrometry Sequencing.

Return to Protocols Used at NCI