Implicaciones del enfoque por competencias para el currículo universitario.,

2.4.1 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS- PAGE)

Use was made of the discontinuous system of SDS-PAGE which consists of two gels, one resolving and one stacking. When cast, the two gels have different porosities, pH and ionic strength. In addition different mobile ions are used in the

gel and electrode buffers (Laemmli, 1970). The buffer discontinuity acts to

concentrate the large volume of sample in the stacking gel, resulting in improved resolution. Proteins, once concentrated in the stacking gel, are separated in the resolving gel on the basis of their molecular masses. The molecular masses of the proteins were determined by use of Ferguson plots. 12% or 15% (v/v) gels were used and proteins separated in the resulting gel were stained with either Coomassie Colloidal blue (Sigma) or silver stain (Pierce).

To visualise separation of SAEP proteins following fractionation on the cation/anion exchange columns, 15% (v/v) gels were used to improve the resolution.

Ch a p t e r 2 G e n e r a l m e t h o d s

The reagents used in the preparation of the SDS PAGE gels were as follows:

♦ Resolving gel buffer was 1.5M Tris-HCI (pH 8.8), stored at 4°C

♦ Stacking gel buffer was 0.5M Tris-HCI (pH 6.8), stored at 4°C

♦ Electrophoresis buffer (x5) was 0.125M Tris-HCI, 0.96M glycine, 0.5% SDS (pH 8.3)

♦ 30% acrylamide - 29.2% (w/v) acrylamide, 0.8% (w/v) bisacrylamide (37.5 : 1) (National Diagnostics, UK).

♦ Ammonium persulphate (APS) 10% solution, prepared fresh (Sigma) with each preparation. APS was added to induce polymerisation of the gels.

♦ TEMED (N'N'-tetramethylethylenediamine) (Sigma). 4pl/10ml of gel buffer ♦ Reducing sample buffer x 4 (Pierce & Warriner (UK) Ltd)

♦ For a 12% (10ml) resolving gel the components were: 4ml 30% acrylamide,

3.3ml HzO, 2.5ml 1.5M Tris-HCI, pH 8.8, lOOpI 10% SDS, lOpI TEMED, lOOpI

10% APS

♦ The stacking gel mixture (5ml) was composed of: 3.4ml H2O 0.83ml 30%

acrylamide, 0.63ml 0.5M Tris-HCI, pH 6.8, 50pl 0.1% SDS, lOpI TEMED,

lOOpI 10% APS

Procedure

1. SDS-PAGE was performed using the Bio Rad Mini Protein II system with 0.75mm or 1mm spacers. The plates were set on the mounting stand. The big glass plate was separated from the small glass plate by the spacers and this sandwich was screwed onto the clamp.

3. 300-400|il of butanol-saturated water was laid on top of the resolving gel to exclude oxygen and prevent the gel surface drying.

4. The stacking gel was added to the top of the plates and the comb carefully inserted at an angle to prevent air bubbles. Once the gel was set, the comb was carefully removed, the wells washed with buffer and the samples loaded. 5. Samples were boiled for 5 minutes in commercial reducing sample buffer

(Pierce & Warriner (UK) Ltd) at 4 part sample: 1 part sample buffer, prior to loading them on the gel. Usually 15-30 pi of sample was loaded into each well with a Hamilton microtitre syringe.

6. The gel was run at 60 amps (per pair) into the stacking gel and at 30 amps

(per pair) into the resolving gel.

2.4.2 2-D gel electrophoresis

To visualise the proteins within SAEP in two dimensions, 2 dimensional gel electrophoresis was carried out, using the Protean II XI multi cell 2-D gel system.

The following reagents were used.

♦ Acrylamide/bis-acrylamide - 146g acrylamide and 4g N'N'-bis-methylene- acrylamide in 500ml water. Stored in dark.

♦ 1.5M Tris-HCI pH 8 . 8 - 91g Tris base in 300ml water, adjusted to pH 8 . 8 with

HOI and made up to 500ml. Stored at 4°C

♦ 0.5M Tris-HCI pH 6 . 8 - 30.5g Tris base in 300ml water, adjusted to pH 6 . 8 with

HCI and made up to 500ml. Stored at 4°C.

♦ Sample solution A - 1g SDS, 0.232g dithiothreitol (DTT), dissolved in 10ml. Aliquoted and stored at -70°C.

C H A P T E R 2 1 1 3

♦ Isourea solution - 0.1 g DTT, 0.2g CHAPS, 5.4g urea, 5 0 0 )liI Bio-Lyte 3-10

ampholyte, 50pl 0.5% bromophenol blue and 6ml distilled water. Aliquoted and

stored at -70°C

♦ Transfer solution - 4ml 0.5M Tris-HCI, pH 8.8, 8ml 10% SDS, 0.8ml 0.05%

bromophenol blue, 15ml water. Stored at 4°C. ♦ Buffers

♦ Upper buffer - first dimension - 0.4g NaOH in 500ml water

♦ Lower buffer - 2.64ml H3PO4 in 41 water

♦ Upper buffer - second dimension (x5) - lOg SDS, 60g Tris base, 288g glycine. Made up to 21.

♦ Lower buffer - lOg SDS 60g Tris base, 288g glycine, 2g sodium azide. Made up to 21.

♦ Detergent solution - 0.3g of CHAPS in 900pl distilled water, lOOpI Nonidet P-40 (in making the detergent solution, CHAPS dissolved in distilled water first and the Nonidet P-40 is added subsequently)

♦ Gel monomer - 5.5g urea, 1.5ml acrylamide/bisacrylamide stock, 0.1ml Bio-Lyte 5-7 ampholyte, 0.4ml Bio-Lyte 3-10 ampholyte and 3ml distilled water.

♦ Second dimension resolving gel solution - (for four gels) 53.5ml of distilled

water, 47.5ml 1.5M Tris-HCI, pH 8 . 8 and 77.5ml of acrylamide /

bisacrylamide stock solution. The 1.5M Tris HCI pH 8 . 8 solution was made

by dissolving 91 g Tris base in 300ml water, adjusting the pH to 8 . 8 with

♦ Second dimension stacking gel monomer - 18.3ml distilled water, 7.5ml

0.5M Tris HCI, pH 6.8, SOOpl 10% SDS, 3.9ml acrylamide/bisacrylamide

stock solution. The 0.5M Tris HCI, pH 6.8, was made by dissolving 30.5g

Tris base in 300ml water, adjusting the pH to 6 . 8 with HCI and making up to

500ml. Stored at 4°C.

Procedure

2 D gel electrophoresis was performed using a Protean II XI multi cell system 1. Separation of the first dimension was by isoelectric focusing. Capillary tube

gel monomer was prepared, avoiding extremes of temperature. 0.5ml detergent solution was added to make the final volume 10ml. The monomer solution was now degassed for 30 minutes. lOpI of TEMED and 20pl of 10% (w/v) APS was added. The capillary tubes were now filled to a previously marked level (13.5cm from one end).

2. Both first dimension buffers were degassed for 1 hour, the lower buffer in the 2D gel tank provided. SAEP (in phosphate buffered saline) at 1 mg/ml was dissolved in solution A in a ratio of 9:1 (v/v) (usually 900|il SAEP + 100|il solution A). The SAEP / sample A mixture was boiled for 5 minutes, cooled at

room temperature and 1|il isourea solution added for every 1 0|il of sample.

Samples were then loaded onto the capillary tubes, overlaid with upper buffer, which filled the upper part of the 2-D gel system, giving sufficient cover for the capillary tubes. The gels were run at 200 volts for two hours, 500 volts for two hours and finally 800 volts overnight (18-20 hours).

3. For the second dimension gels, casting chambers were assembled, as instructed with the Protean II XI system. The second dimension resolving gel

C H A P T E R 2 1 1 5

monomer was prepared and degassed for 30 minutes, then 44pl TEMED and 438|il of 10% (w/v) APS were added. The gels were poured and covered with butanol and cling film. The gels were allowed to set overnight at 4°C. The stacking gels were poured after removing the butanol layer and adding ISOpI of 10% APS, 30pl of TEMED to the stacking gel monomer (volume 30ml).

4. The first dimension tube gels were extruded from the capillary tubes onto plastic sheets filled with transfer buffer and then laid on top of the second dimension stacking gel ensuring no bubbles intervened between tube gel and slab gel. 20pl Dalton VII markers (Sigma) were prepared by heating to 100°C for 5 minutes and loaded onto the gel. The upper chamber was filled with second dimension upper reservoir buffer, to 5mm below the outer glass plates. The gels were run at 35mA per gel. Electrophoresis took approximately 3-4 hours.

5. When electrophoresis was complete, each slab gel was removed and fixed overnight in 45% methanol and 10% acetic acid solution. The gels were then stained with Coomassie Brilliant Blue. The stain was made up using 45% methanol, 10% acetic acid and 0.25% Coomassie stain (w/v). De-stain was performed with 45% methanol and 10% acetic acid.