Biochemical Methods

2.4.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE)

The SDS-PAGE is based on the ability of SDS, an anionic detergent, to denaturate and charge uniformly negatively protein molecules. Thus, the proteins undergoing an electrophoresis are separated only according to their molecular weight. For better sample resolution, a discontinuous SDS-PAGE was performed according to Laemmli (1970).

SDS-PAGE was performed using SE 600 vertical slab gel unit, 1.5 mm combs and spacers, 180x140 mm glass plates, and EPS 1001 power supply (AmershamPharmaciaBiotech).

Acrylamide solution 30% (w/v) acrylamide, 0.8% bisacrylamide 4x Running gel buffer 1.5 M Tris/HCl (pH 6.8), 0.4% (w/v) SDS 4x Stacking gel buffer 0.5 M Tris/HCl (pH 8.8), 0.4% (w/v) SDS Ammonium persulfate 10% (w/v) APS

TEMED

2x Sample buffer 250 mM Tris/HCl (pH 6.8), 2% (w/v) SDS, 20%

(w/v) glycerol, 20 mM DTT (only for reducing conditions)

Anode buffer 192 mM glycine, 50 mM Tris/HCl (pH 8.6)

Cathode buffer 192 mM glycine, 50 mM Tris/HCl (pH 8.6), 0.1%

(w/v) SDS, 0.001% (w/v) bromphenol blue

Discontinuous electrophoresis consisted of a separating gel (5-15% acrylamide, 0.375 M Tris/HCl pH 8.8, 0.1% SDS, 0.8% APS, 0.08% TEMED) and stacking gel (4% acrylamide, 0.125 M Tris/HCl pH 6.8, 0.1% SDS, 0.33% APS, 0.1% TEMED). The separating gel solution was carefully pipetted down between the glass plates, overlayed with ddH2O and allowed to polymerize at room temperature for 30 min. After pouring off the overlaying ddH2O, the stacking gel solution was pipetted down and a comb was inserted. The gel was allowed to polymerize for 60 min. The comb was removed and wells were rinsed with ddH2O, which was discarded by aspiration. Probes solubilized in a sample buffer, and boiled at 95°C for 5 min, were loaded on the gel and overlaid with cathode buffer.

Prestained molecular weight markers (RPN 756, low range 14,300-220,000, Amersham Pharmacia Biotech) were used as standards. Electrophoresis was run at 50 mA/gel in a chamber filled with anode buffer, typically for 2-3 h.

2.4.2 Detection of proteins in SDS-polyacrylamide gels

2.4.2.1 Coomassie Staining

After SDS-PAGE, the gel was simultaneously fixed and stained in 50% methanol, 10%

acetic acid and Coomassie blue R250 at room temperature for 30 min. The gel was destained in 50% methanol, 10% acetic acid, washed with ddH2O, and dried between celophane sheets with an air gel dryer (BioRad, Munich, Germany).

2.4.2.2 Silver staining

(according to Blum et al., 1987)

Gel was fixed in 40% (v/v) ethanol and 10% (v/v) acetic acid for 60 min. Washing with 30% (v/v) ethanol (3x20 min) was followed by an incubation in 0.02% (w/v) sodium thiosulfate for exactly 1 min. The gel was washed in ddH2O (3x 20 sec) and stained in 0.2% (w/v) silver nitrate, and formaldehyde (250 µl 35% formaldehyde per 1 l) for 20 min.

Protein bands were developed in 3% (w/v) Na2CO3 and formaldehyde (500 µl 35%

formaldehyde per 1 l) for 2-5 min. The reaction was terminated in 1% (w/v) glycine for 10 min, the gel was washed in ddH2O, and subsequently air-dried.

2.4.3 Protein transfer from SDS-PAGE gels to membranes

Protein electrotransfer from SDS-gels to nitrocellulose membranes was carried out according to Towbin et al. (1979).

Transfer buffer 25 mM Tris/HCl, 192 mM Glycine, 20% Methanol (v/v) Separating gel, nitrocellulose membrane (0.2 µm pore size, Sartorius, Germany), 2 sponges and 4 sheets of 3MM Whatman paper were equilibrated in transfer buffer for 5 min. A transfer sandwich (sponge, 2 sheets of paper, nitrocellulose membrane, running gel, 2 sheets of paper, sponge) kept together by a plastic cassette, was assembled under transfer buffer to minimize trapping of air bubbles. The cassette was inserted in the buffer tank and the transfer was run at 900 mA for 90 min in TE 62X and 400 mA for 1 h in TE 22 unit

(Tank Transphor TE 62X or TE 22 Mighty Small Transphor, Hoefer Scientific Instruments, San Francisco, USA).

2.4.4 Detection of proteins on nitrocellulose membranes 2.4.4.1 Western immunoblot

PBST 0.1% (w/v) Tween 20 in PBS

Blotto 5% (w/v) fat-free milk powder in PBST

Membranes were blocked for 1 h at room temperature in Blotto. The blots were incubated with rocking for 2 h at room temperature with the primary antibodies (Table 3) diluted in Blotto. The membranes were rinsed five times with PBST and the respective secondary antibody (Table 4) was added. After incubation for 1 h at room temperature, the blots were rinsed as before and the antibody binding was visualised with ECL kit (SuperSignal, Pierce, Rockford, IL) by means of X-Omat AR films.

Primary Antibody Host Dilution Producer

Anti-human IGFPB-1 rabbit 1:1000 UBI, Lake Placid, NY Anti-bovine IGFBP-2 rabbit 1:1000 UBI, Lake Placid, NY Anti-human IGFBP-4 rabbit 1:1000 UBI, Lake Placid, NY Anti-human IGFBP-5 rabbit 1:1000 UBI, Lake Placid, NY Anti-human IGFBP-6 rabbit 1:600 van Doorn et al., 1999

Anti-mouse IGFBP-6 goat 1:450 Santa Cruz Biotechnology, Inc.

Anti-human ADAM-12

disintegrin domain (rb 119) rabbit 1:500 Dr. U. Wewer¹ Anti-human ADAM-12

cysteine-rich domain (rb 122) rabbit 1:500 Dr. U. Wewer² Anti-human ADAM-12

prodomain (rb 132) rabbit 1:500 Dr. U. Wewer²

Table 3: Primary antibodies used in Western immunoblotting

1 The antibody was kindly provided by Dr. U. Wewer, University of Copenhagen, Denmark.

Secondary Antibody Dilution Producer

Peroxidase-conjugated rabbit anti goat IgG 1:7000 Jackson Immuno Researach Lab., Inc.

Peroxidase-conjugated goat anti rabbit IgG 1:7000 Jackson Immuno Researach Lab., Inc.

Table 4: Secondary antibodies used in Western immunoblotting

2.4.4.2 Western ligand blot (WLB)

For detection of target protein(s) in Western ligand blotting, a labeled ligand is used instead of a primary antibody. For a successful protein-ligand binding, preservation of the secondary protein structure is necessary, which requires exclusion of reducing agents from the SDS-PAGE.

For detection of IGFBPs, biotin- or [¹²⁵I]-labeled IGF I or II were used.

2.4.4.2.1 Biotinylated IGF I and IGF II (bIGF) WLB

bIGF I and bIGF II, and streptavidin-HRP were purchased from GroPep, Adelaide, Australia and Jackson Immuno Research Laboratories, Inc., respectively.

PBT 8 mM Na2HPO4, 2 mM NaH2PO4, 10 mM NaCl, pH 7.5, 0.1% Tween 20

BSA-PBT 1% (w/v) BSA in PBT

Membranes were blocked in BSA-PBT for 1 h at room temperature and incubated with bIGF II (20 ng/ml in BSA-PBT) for 16 h at 4°C. The blots were rinsed five times with PBT and incubated with streptavidin-HRP/BSA-PBT (1:7,000) for 45 min at room temperature.

The membranes were washed as mentioned before, and the proteins detected by ECL, as described earlier (2.4.4.2).