SDS-polyacrylamide-gel electrophoresis and western blotting

In the general procedure, SDS-polyacylamide-gel electrophoresis was performed according to Laemmli (Laemmli, 1970).

Separating gel Lammeli gel components

10% 12%

Stacking gel

30% Acrylamide/Bis (37.5: 1) 2.6ml 3.2 ml 540µl

H₂O 3.6ml 3ml 1.1ml

TGP 5x (1.8M Tris, 7mM SDS pH 8.8) 1.6ml 1.6ml

-SGP 2x (0.25M Tris, 7mM SDS pH 6.8) - - 1.6ml

10% APS 66µl 66µl 33µl

TEMED 16µl 16µl 6µl

For the analysis of nuclear pore proteins, SDS-PAGE was performed according to Thomas and Kornberg (Thomas and Kornberg, 1975).

Separating gel

For optimal resolution of proteins of a given target size, an appropriate acrylamide content was selected for the separating gel, while the percentage of acrylamide of the stacking gel remained constant. APS and TEMED were added to initiate the polymerisation reaction.

All samples were prepared in 1x running buffer (Lammeli 10x: 250mM Tris, 1.92M glycine, 1%SDS; Thomas Kornberg 5X: 0.05M Tris, 0.38M glycine, 0.1% SDS) and boiled with 0.1x volume sample buffer (584mM Tris pH 6,8, 17% SDS, 60% glycerine, 1.43M -mercaptoethanol) at 95°C for 5 min and loaded in equal volumes. Gels were run at a constant voltage of 40V until the samples left the stacking gel. Thereafter the voltage was gradually increased to 100V.

MATERIALS AND METHODS

After electrophoresis was completed, six layers of Whatman 3MM paper and nitocellulose membrane were all cut to the size of the gel and soaked in blotting buffer. The blotting sandwich was prepared as follows: one pre-soaked fibre pad was placed on the cathode side of the sandwich cassette, followed by 3 layers of Whatman 3MM paper, the gel, followed by the nitrocellulose membrane, 3 layers of Whatman 3MM paper and finally another fibre pad. Air bubbles between the layers were removed by rolling a pasteur pipette across the stack. Proteins were transferred at a constant current of 400mA for 1h, 0.03% SDS was then added on the cathode side of the tank blot apparatus and transfer was allowed to continue at 4°C for 18-24h at 350mA.

After immunoblotting, the protein transfer and loading efficiency was assessed by staining the membrane with Ponceau red (0.2 % Ponceau S, 5 % acetic acid). The membranes were destained by several washings in water and finally in TNT (150mM NaCl, 50mM Tris/HCl pH 8, 0.05% Tween 20). The membrane was incubated in TNT plus 5% non-fat dry milk overnight at 4°C to block non-specific binding of the antibody. After removal of excess milk protein by extensive washings in tap water the membrane was equilibrated in TNT.

Incubation with the primary antibody was performed in a volume of 5 ml 5% milk/TNT overnight at 4°C. Non-bound antibody was removed by washing the membrane in TNT (1x 15 min, 2 x 5 min) prior to incubation for 1h with the secondary horseradish peroxidase (HRP)-conjugated antibody.

Detailed protocol for immunodetection of:

Nucleoporin 50

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:500 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-rabbit HRP secondary antibody in a dilution of 1:1500 in 5% milk/TNT for 1h at room temperature.

Nucleoporin 93-3, Nucleoporin 96-1 and Nucleoporin 98-2

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:130 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-guinea pig HRP secondary antibody in a dilution of 1:1500 in 5% milk/TNT for 1h at room temperature.

Nucleoporin 107-1 and Nucleoporin 205-2

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:130 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-guinea pig HRP secondary antibody in a dilution of 1:1500 in 5% milk/TNT for 1h at room temperature.

Nucleoporin 133

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:2500 in 5% milk/TNT overnight at 4°C, followed by incubation with affinity purified goat anti-rabbit HRP secondary antibody in a dilution of 1:2000 in 5% milk/TNT for 1h at room temperature.

Nucleoporin 153

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:8 in 5% milk/TNT overnight at 4°C, followed by incubation with affinity purified goat anti-mouse HRP secondary antibody in a dilution of 1:1000 in 5% milk/TNT for 1h at room temperature.

Nucleoporin160

80µg protein/lane was loaded on a 10% gel. Primary antibody was used in a dilution of 1:500 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-rabbit HRP secondary antibody in a dilution of 1:2000 in 5% milk/TNT for 1h at room temperature.

Nucleoporin 188

80µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:500 in 5% milk/TNT overnight at 4°C, followed by incubation with affinity purified goat anti-rabbit HRP secondary antibody in a dilution of 1:2000 in 5% milk/TNT for 1h at room temperature.

Nucleoporin 50

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:500 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-rabbit HRP secondary antibody in a dilution of 1:2000 in 5% milk/TNT for 1h at room temperature.

MATERIALS AND METHODS

RanBP2

For RanBP2 detection, 80µg protein/lane was loaded on a 10% gel. The primary antibody was used in a dilution of 1:1000 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-guinea pig HRP secondary antibody in a dilution of 1:1500 in 5% milk/TNT for 1h at room temperature.

Tpr

80µg protein/lane was loaded on a 10% gel. The primary antibody was used in a dilution of 1:130 in 5% milk/TNT overnight at 4°C, followed by incubation with a goat anti-mouse HRP secondary antibody in a dilution of 1:1000 in 5% milk/TNT for 1h at room temperature.

CAN/Nucleoporin 214

80µg protein/lane was loaded on a 10% gel. Primary antibody was used in a dilution of 1:200 in 5% milk/TNT overnight at 4°C, followed by incubation with a goat anti-mouse HRP secondary antibody in a dilution of 1:1000 in 5% milk/TNT for 1h at room temperature.

Actin

The membrane was re-probed with anti-actin antibody in a dilution of 1:10 000 in 5%

milk/TNT for 1h, followed by incubation with goat anti-mouse HRP secondary antibody in a dilution of 1:1000 in 5% milk/TNT for 1h at room temperature.

Caspase-3 (cleaved fragments)

The membrane was re-probed with anti-caspase-3 in a dlution of 1:1000 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-rabbit HRP econdary antibody in a dilution of 1:2000 in 5% milk/TNT for 1h at room temperature.

Lamin A/C

For the detection of lamin A/C, 40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:50 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-mouset HRP secondary antibody, in a dilution of 1:1000 in 5%

milk/TNT for 1h at room temperature.

Lamin B

For the detection of lamin B, 40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:500 in 5% milk/TNT overnight at 4°C, followed by incubation with donkey anti-goat HRP secondary antibody, in a dilution of 1:2000 in 5%

milk/TNT for 1h at room temperature.

Parp

40µg protein/lane was loaded on a 12% gel. Primary antibody was used in a dilution of 1:5000 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-mouse HRP secondary antibody, in a dilution of 1:1000 in 5% milk/TNT for 1h at room temperature.

RanGAP

80µg protein/lane was loaded on a 10% gel. Primary antibody was used in a dilution of 1:1000 in 5% milk/TNT overnight at 4°C, followed by incubation with goat anti-rabbit HRP secondary antibody in a dilution of 1:2000 in 5% milk/TNT for 1h at room temperature.

After another three washing steps, HRP activity was detected by enhanced chemiluminescence (ECL). The membrane was soaked for 5 min in the detection reagent.

This elicits a peroxidase-catalysed oxidation of luminol and subsequently enhanced chemiluminescence, where the HRP labelled protein is bound to the antigen on the membrane. HRP activity was visualised by exposure to x-ray films or recorded in a luminescence image analyser.