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.

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

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.