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2.9.1 Isolation of Nuclear and Cytosol Extracts

All buffers and reagents used for the isolation of nuclear and cytosol extracts were chilled on ice before commencing the protocol. The following volumes are appropriate for up to 1.25 x 107 cells.

Cells were spun down at 500g for 5 minutes at 4oC, then the supernatant removed by aspiration. The side of the tube was gently tapped to loosen the cell pellet, which was then resuspended in 1 ml ice-cold PBS. A further 9 ml PBS was added, then the were spun down again at 500g for 5 minutes at 4 oC. The supernatant was removed and the cell pellet placed on ice. Again, the pellet was loosened, then resuspended in 1 ml of Buffer A containing Igepal, and transferred to an Eppendorf tube and incubated on ice for 5 minutes. The tubes were then spun at 1500g for 5 minutes at 4oC. The supernatant was removed and transferred to a clean Eppendorf tube. These cytoplasmic extracts were stored at –80oC until required.

The pellet was resuspended in 1 ml Buffer A (no Igepal), then spun for 5 minutes at 1500g. During this spin, 20μl of Protease Inhibitor Cocktail (Sigma) and

1μl of dithiorithrol (Bio-Rad) were added to 1 ml of Buffer C. After spinning, the supernatant was removed and the pellet resuspended in 25μl of Buffer C. This was

incubated on ice with shaking for 15 minutes at 4oC, then spun at 13,000g for 5 minutes at 4oC. The supernatant was removed to a clean tube, and these nuclear extracts were stored at – 80oC until required.

Protein content of these samples was assayed in duplicate using Bradford reagent (Bio-Rad) at 490 µm.

2.9.2 Polyacrylamide Gel Electrophoresis (PAGE)

Proteins were separated by polyacrylamide gel electrophoresis (PAGE). For proteins in the size range of 10-100 kDa, 12 % gels were used. 12% separating gels were cast into plastic cassettes (Invitrogen) and overlayed with 1 ml saturated butanol solution (butyl-alcohol plus dH2O) until set (approximately 20 minutes). After setting, the butanol solution was removed, the cassette was washed with dH2O, and the stacking gel poured over the separating gel, the comb was inserted, and all bubbles removed.

50μg of cytosolic samples, and 30 μg of nuclear samples were run per lane.

Prior to loading, samples were mixed with 3x Laemmli sample loading buffer and boiled for 5 minutes.

The cassette was placed into a Novex XCell II mini cell (Invitrogen), the cell filled with running buffer, and the comb removed. Samples and Protein-Pure ladder (Bio-Rad) were loaded, then run at 30 mA until sufficient separation of the ladder dye bands was observed.

2.9.3 Electro-Blotting

Samples were electroblotted onto nitrocelullose membranes (Amersham or Bio- Rad). the membrane and filter papers were trimmed to the size of the gel, and pre-wet in a shallow dish of electro-blot buffer. Gels were carefully removed from the cassette and layered with a blotting pad on the bottom (negative side), and on top of this, in order, a filter paper, the gel, the nitrocellulose membrane, another filter paper, and finally, another blotting pad. All bubbles were carefully removed from the sandwich before assembling blot module in the dH2O-rinsed Novex XCell II mini cell (Invitrogen). The cell was filled with electroblot buffer, completely submerging the sandwich, and then the blot was run overnight at 30 V and 4oC.

2.9.4 Antibody Staining

After electroblotting, membranes were removed from electroblot buffer and blocked with 5% low-fat skim-milk powder in TBS-Tween for 30 minutes at room temperature with rocking. Primary antibody was applied at a suitable dilution (eg.

1:1000) in 5% milk powder in TBS-Tween, and incubated at room temperature for 1 hour with rocking. Membranes were washed four times in TBS-Tween for 5 minutes each at room temperature with rocking. Secondary antibody was applied at 1:5000 in 5% milk powder in TBS-Tween at room temperature for 1 hour with rocking. Membranes were again washed four times in TBS-Tween. The membrane was kept in TBS-Tween to prevent drying out.

2.9.5 Enhanced Chemi-Luminescent Detection

Washed membranes were incubated in a 1:1 mixture of ECL Buffer and ECL Reagent (Sigma) for one minute. Membranes were exposed to X-ray films (Amersham) in a light proof container, in darkroom conditions. Timing of exposure was adjusted as appropriate.

Films were developed in Kodak Polymax RT Developer and Replenisher (Sigma), washed ten times briefly in running tap water, and then fixed for 10 minutes in Kodak Polymax RT Fixer and Replenisher (Sigma). Films were then washed for a minimum of 10 minutes in running tap water before air-drying.

2.9.6 Semi-Quantitative Analysis of Western Blots

Films of the Western Blots were scanned into the Quantity One program (Bio- Rad) and the peak intensity of the bands representing the fusion protein and the β-actin loading control were measured. To account for minor differences in the amount of protein loaded, the staining intensity of the fusion protein was expressed as a proportion of the staining intensity of the loading control. This analysis is considered to be semi- quantitative.

2.9.7 Cell Proliferation and Viability Assays

Cell proliferation assays were performed with a colorimetric ‘Cell Titer 96’ cell proliferation assay (Promega). 10 µl of ‘Cell Titer 96’ reagent was added to 200 µl of treated cells in a flat-bottom 96-well plate. The plate was returned to the incubator for 2 hours, and then absorbance was read at 490 nm using a Spectra max M2 plate reader, and SoftMax Pro 4.2 software (Molecular Devices, Surrey Hills, Australia). Raw

absorbance was converted to total cell number using a set of standard curves for each cell type.

2.9.8 Statistical Analyses

For many of the preliminary studies, where the sample size was less than three, statistical analysis was not performed. For the qRT-PCR and cell proliferation assays, there was considerable variation between replicate data sets, and analysis consisted of paired, one-tailed t-tests.