REGISTROS DE DESPLAZAMIENTO

2.2.5.1 Sequencing Reaction

The dRhodamine terminator cycle sequencing chemistry kit (Perkin Elmer AppUed Biosystems, UK) was used to perform the sequencing reactions. NormaUy, the 10 pi sequencing reactions were carried out. To each 0.5 ml tube, 0.5 pg o f plasmid DNA fi*om mini prepare was added, 4 pi o f dye terminator premix and 1 pi o f 1.6 pmol/pl primer was added and the final volume was made up to 1 0 pi with sterUe

water. The mixture was votexed, centrifuged, overlaid with 20 pi o f mineral oU and placed on a preheated (96 °C) PCR machine. The PE-ABI DNA thermal cycler model 480 and the cycle conditions were employed as recommended by the manufacturers.

2.2.5.2 Purification of Sequencing Products

The sequencing products were taken from the reaction tube after being diluted with 10 pi o f dHzO, and incubated with 300 pi o f 95% ethanol and 10 pi o f 3 M NaOAc (pH 5.2) at RT for an additional 15 minutes. The mixture was then spun at 13,000 rpm at desktop centrifiige for 20 minutes. The supernatant was removed and the resulting invisible pellets were washed twice with 200 pi o f 70% ethanol by briefly votexing and spinning. The final pellets were placed on a PCR block for 5 minutes at 80 °C to remove all traces o f ethanol and stored either at room temperature for a day or -20 °C for longer periods.

2.2.5.3 Preparation of Sequencing Gel and Loading of Samples

3 pi o f formamide loading buffer was added to the samples (dextran sulphate at 30 mg/ml in a ratio o f 5:1 (v/v) 25 mM EDTA (pH 8.0) to deionised 98% formamide, 10 mmol/L EDTA), denatured for 2 minutes at 96 °C and immediately placed on ice, 2 pi o f the product was loaded onto a sequencing gel.

2.2 5.4. Sequencing Analysis

Sequencing analysis was carried out using Sequencing analysis 2.0 (PE-ABI, UK) programme.

2,2,6 Protein Analysis

2.2.6.1 Protein Analysis by Sodium Dodecyl Sulphate (SDS) -Polyacrylamide Gel Electrophoresis (PAGE)

The recombinant apoCIII proteins with MW ranging from 12-43 kDa, including glutathione S-transferase (GST)-apoCIII fusion protein, S-protein fusion apoCIII (see detail in Chapter 5) and apoE were separated on 15% SDS-PAGE

according to described by Maniatis et al (Maniatis T et al., 1989), using Pharmacia (Uppsala, Sweden) system according to manufecturer’s instruction.

The 6xHis-tag fused apoCIII proteins (both C-terminal and N-terminal fusion

proteins) were separated on 17.5 % precast Tricine-SDS-PAGE (separation range MW 26.6-1.4 kDa) provided by Bio-Rad (Hertfordshire, UK).

2.2.6.2 Coomassie Blue Staining of Polyacrylamide Gels

Coomassie blue staining was performed according to described by Maniatis et al. (Maniatis T et al., 1989). Gels were immersed in at least 5 volumes o f a concentrated solution o f the dye (0.5% Coomassie Brilliant Blue in 10% HAC, 30% isopropanol) and placed on a slowly rotating platform at room temperature for at least 30 minutes. The gels were then destained in 30% methanol, 10% HAC solution as described above. The excess dye was then allowed to difiuse from the gel during a prolonged period o f destaining as long as required for the appearance o f the bands. The gels were rinsed with distilled water and fixed in distilled water containing 20% glycerol prior to dry.

2 2.6.3 Silver Staining of Polyacrylamide Gels

Visualisation o f protein on polyacrylamide gels was achieved by the method o f Merril (1981). All solutions except developer could be kept at room ten^erature for months. Gels were fixed by soak in 50% methanol, 10% HAC for 20 minutes followed by soak in 5% MeOH and HAC for 20 minutes. After washed in distilled water for 10 minutes, gels were soaked in 10 pg/ml DTT for 20 minutes then immersed in 0.1% AgNO] for 20 minutes and washed with distilled water briefly. Precipitation o f silver was achieved by first rinsing gels in freshly made developer (30 g NazCOg, 0.5 ml formaldehyde per litre) until the solution turned yellow-brown (30-40 seconds) and then soaking in the developer as long as required for the appearance o f the bands. Finally the reaction was stopped by adding HAC. The gels were then rinsed well with distilled water and fixed in distilled water containing 2 0% glycerol prior to dry.

2 2.6.4 Western-blotting

Protocol 1

The separated proteins were transferred onto a nitrocellulose membrane using a Pharmacia transblotting device and then the membrane was put in TBST buffer containing 3% BSA for 30 minutes at room temperature. After 2 hours incubation with a rabbit-anti apoCIII polyclonal antibody (1:100 dilution in BSA/TBST), the membrane was washed for 3x15 minutes in TBST. Then the membrane was incubated with anti-rabbit IgG alkaline phosphatase conjugate (1:20000 in BSA/TBST) for 1 hour. Finally the membrane was washed three times for 5 minutes in TBST. ApoCIII protein was visible in 100 mM Tris-HCl (pH 9.5), 100 mM NaCl, 5 mM MgCl2

containing 0.4 mM nitroblue tétrazolium (NBT) and 0.4 mM 5-bromo-4-chloro-3- indolylphoshate (BCIP) The reaction was stopped by rinsing the membrane in 20 mM Tris-HCl (pH 8.0) containing 5 mM EDTA.

Protocol 2

The separated proteins were transferred onto a Immobilon membrane (Bio- Rad) using a semi-dry blotting apparatus (Bio-Rad) in a Tris (5mM)-Glycine (192 mM) buffer at pH 8.3 containing 20% methanol. Proteins were visualised with a 1:100 dilution o f polyclonal antibodies o f rabbit-anti apoCIII (for apoCIII detection), or a rabbit-anti apoE (for apoE detection), and a secondary peroxidase labelled goat anti­ rabbit antibody (Sigma). The process was performed similarly to described in Protocol 2, apart from using 1% dry milk powder in phosphate buffer saline (PBS, pH 7.5) containing 0.02% Tween-20 instead o f the TBST buffer containing 3% BSA. The bound peroxidase was visualised using the BM Chemiluminescence luminal substrate provided by CN-DAB kit (Pierce).