2D PAGE

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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 aggregatesshould 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 g/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 m to separate epithelial cells from stroma and filtered through a

nylon mesh with 200 m 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 l per106 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 l of the

final diluted colon epithelial cell sample was loaded onto the IPG gel strip.

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

with 500 l of ice cold acetone and centrifuged at 10000 g at 4o C for 10

minutes. The pellet was mixed with 10 l of a solution containing SDS (10%

w/v) and DTE (2.3% w/v). The sample was heated to 95 o C for 5 minutes

and then diluted to 60 l 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 g) 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

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l 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 andremoval of DMEM, 0.8 x 106 cells were mixed and solubilized with 60 l 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

l in a MicrosepTM Concentrators. The concentrated sample was mixed

with 60 l 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 finaldiluted 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 bysquirting 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 l of a solution containing urea (8 M), CHAPS (4%


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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 g/ml, gentamycin 50 g/ml, penicillin 100 U/ml, streptomycin

100 g/ml, amphotericin B 0.25 g/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 l 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 l of the final

diluted kidney cell sample was loaded onto the IPG gel strip.

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

liver biopsy were mixed with 300 l of a solution containing urea (8 M),

CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of bromophenol

blue. Sixty l (45 g) 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 l (100

g) of the final diluted liver sample was loaded on the first dimensional

separation.

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

lymphoma biopsy were mixed with 300 l of a solution containing urea (8

M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and a trace of

bromophenol blue. Sixty l (45 g) 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
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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 l of human plasma was mixed with 10 l 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 l with a solution

containing urea (8 M), CHAPS (4% w/v), Tris (40 mM), DTE (65 mM) and

a trace of bromophenol blue. Sixty l (45 g) 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. Thesupernatant (platelet-rich plasma: RPR) was then centrifuged at 5000 g for

10 minutes. 106 washed platelets were suspended in 500 l 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 l in aMicrosepTM Concentrator. The concentrated protein sample was mixed and



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 l of RBC wasmixed with 483 l of a solution containing urea (8 M), CHAPS (4% w/v),

Tris (40 mM), DTE (65 mM) and a trace of bromophenol blue. Forty l (45

g) of the final diluted RBC sample was loaded on the first dimensional

separation [4].
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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 l 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 g of dried brown adipose tissue

was mixed with 60 l 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 g) was loaded in acup at the cathodic end of the IPG gels.

ISLETS (Pancreatic islet cells): Two hundred of small and large pancreatic

islets were mixed with 60 l 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 g) was loaded in a

cup at the cathodic end of the IPG gels.

LIVER: Two hundred g of dried liver was mixed with 60 l 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 g) 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 g of dried gastrocnemius

muscle was mixed with 60 l 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



WAT (White adipose tissue): Sixteen mg of dried white adipose tissue was

mixed with 60 l of a solution containing urea (8 M), CHAPS (4% w/v),

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Tris (40 mM), DTE (65 mM), SDS (0.05% w/v) and a trace of bromophenol

blue. The whole final diluted sample (150 g) 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 l of the sample(" in-gel sample rehydration "), and completed to a total of 450 l 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 l of a solution

containing urea (8 M), CHAPS (4 % w/v), Tris (40 mM), DTE (65 mM) and

a trace of bromophenol blue. Nine l of this sample was diluted with 60 l

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 l 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 l of the latter was mixed with 60 l 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.
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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 m 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 g (measured by

modified Lowry) of yeast proteins was mixed with 60 l 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 (10mM), 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].
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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) andDTE (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:
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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 seconddimension 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 70 o 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
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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).

ScanningThe 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.
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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 operatingroom, 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 g/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 m to separate epithelial cells from stroma and filtered through a

nylon mesh with 200 m 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 wereincubated with fluorescein-conjugated anticytokeratin antibodies (CAM 5.2)

for 45 min and then sorted by FACS. The pellet was mixed with 100 l 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 l of the final diluted colon epithelial cell sample was used for in-

gel sample rehydration.

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

with 6000 l of ice cold acetone and centrifuged at 10000 g at 4 o C for 10

minutes. The pellet was mixed with 500 l of a solution containing urea (8M), 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 l 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

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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 l in a MicrosepTM Concentrators. The concentrated sample wasmixed with 400 l 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


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 g/ml, gentamycin 50 g/ml, penicillin 100 U/ml, streptomycin

100 g/ml, amphotericin B 0.25 g/ml, nistatin 50 U/ml) to further remove

cell debris and blood, and were frozen by immersion in liquid nitrogen. They

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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 l 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

l 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 l 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 andmixed with 500 l 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 m x 5 mm x 10 mm) of a human

lymphoma biopsy were mixed with 500 l 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 containingurea (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 l 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 l in a

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MicrosepTM Concentrator. The concentrated protein sample was mixed and


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 wasgrown 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 l 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 l 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 g) 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 l 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



LIVER: Four mg of dried liver was mixed with 60 l 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 g) was loaded in a cup at the cathodic end of the IPG gels.
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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 l 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



WAT (White adipose tissue): One hundred and sixty mg of dried white

adipose tissue was mixed with 60 l 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 g) wasloaded 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 l 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


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 l 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

-20C. Fifty l of the latter was mixed with 450 l 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.

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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 m 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 l 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


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 ofnarrow 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].

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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].



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 copynumber 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
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"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 g to 15 mg of proteins were solubilized with 500 l 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 werecovered 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.
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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.



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/vSDS, 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 orsemi-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 3min in water and three times in transfer buffer. These two steps are

important in order to avoid any protein or amino acid contamination.
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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 tankwith 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 (13mM), 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 1min 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 tankwith 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/vcathodic side) at 1 mA/cm2 constant current for 3 hours at 15oC

or as described by the manufacturer.
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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


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


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].
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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.
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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 produceseasily 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 onthe 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 heatingto 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.
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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 l 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 l 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 autosamplervials 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 themembranes 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

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(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 l 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 extractionsolution.

4. Place the autosampler vials containing protein hydrolysates in a

vacuum dryer (Savant Speedvac) and evaporate to dryness.

5. Add 10 to 20 l 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 canaccurately manipulate 10 l volumes. We use a GBC Aminomate Amino

Acid Analyzer(GBC Scientific Instruments, Dandenong, Vic., Australia) for

this purpose.
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Derivatisation of amino acids with 9-fluorenylmethyl chloroformate

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

2. Starting with 10 l of hydrolysate, add 10 l of Fmoc reagent and mix

thoroughly by pipetting up and down. Wait for 90 sec.

3. Add 10 l of cleavage reagent and mix thoroughly by pipetting. Wait

for 3 min 30 sec.

4. Add 10 l 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 foreach 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).
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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 blockedat 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
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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.

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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 ourspecific 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

2 g Chaps

0.5 g Mega 10

0.5 g OBG

250 l Triton X-1000.25 g Tris

0.4 g DTT

500 lPharmalytes or IPG buffer pH 3-10

(Amersham)

500 l - mercaptoethanol

4. Add 10 l 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 -20C.

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

glycine, 1M SDS

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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 m pore size filter.

5. Store at 4C 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

e Units

9% gel 18% gel

1.5 M Tris-HCl,

pH 8.8

ml11.5 11.5

20% SDS ml 0.23 0.23

30% Acrylamide ml 14 28

TEMED l 11.7 11.7

10% APS l 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.

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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 IodoacetamideINSTEAD 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 m 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 theremaining xylene.

13.Add 400 l 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.
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To lyse a5-8 m 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 m 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 l 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 l 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 l to the lysates for a total of 400 l.


8. Incubate for 5 min at RT.


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:

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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

-20C 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 mlDryStrip Cover fluid.3. Remove strips.

4. Place on Whatman paper gel-side up.

TIP: Placing the strips gel-side down might result in protein lossand

gel damage.

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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 immobilonstrips and outside the electrodes.

13.Electrophorese for 36-48 hrs, using the following sequence of

settings:

Voltag

eAmps

Wattag

eTime

1 500 V100

mA33 W 0.05 hrs

2 500 V110

mA 70 W 1 hr

3 3500 V141

mA32 W 5 hrs

4 3500 V 70 mA 38 WUntil

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 whicheverside. Continue running until the dye has disappeared.

Day Three: 2nd Dimension

A: Prepare Apparatus

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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 cmMeylar 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.

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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 inchfrom both sides.

11.Place gels with basic side clo

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