CONCEPTO JURÍDICO DE DIVORCIO

2 . 6 . 1 Materials and Solutions

iiron -% extraction buffer

0.5% v/v Triton X-100, 120mM NaCl, 25mM KCl, 2mM CaC12, 15mM Tris-HCl pH

7.5. 2mM phenylmethanesulphonyl fluoride (PMSF), O.lmM dithiothreitiol (DTT) and 1 pg/ml aprotinin were added to the extraction buffer just before addition to the cells. Stock PMSF was a lOOmM solution in acetone, and was stored at 4°C. DTT stock was a O.IM solution in H^O and stored at -20°C. The aprotinin was a 100 pg/ml solution in dH^O and stored at 4°C.

Running buffer

50mM trisma-base, 384mM glycine and 0.1% SDS

Sample buffer

2x laemmh sample buffer (reducing) comprised 125 mM Tris-HCl pH6.8, 2%SDS, 20% glycerol, 0.02% Bromophenol blue in 10% (v/v) P-mercaptoethanol

Ponceau S solution

2% Ponceau S (3-hydroxy-4-[2-sulpho-4-(sulpho-phenylazo]-2,7-naphthalene disulphonic acid) in PBS, 30% Acetic Acid, 30% Sulphosalicylic acid. The stock was diluted 1:10 with water to produce the working solution.

semi-dry transfer buffer

lOx stock solution comprised of 0.2M Tris-HCl, pH7.5 and 1.5M glycine. The solution was stored at room temperature. The Ix solution was made in 0.1% SDS and 20% (v/v) absolute methanol.

Glycine stripping solution

200mM Glycine pH 2.5, 0.4% SDS

Harsh stripping solution

62.5 mM Tris/HCl pH 6.7, 2% SDS, lOOmM p-mercaptoethanol

2 . 6 . 2 Preparation of SDS-PAGE gels

Stock solutions Solution A Solution B Solution C Solution D AP TEMED

30% acrylamide / 0.8% bisacrylamide (Millipore), 4oC 3M Tris-HCl, pHS.S (BDH)

10% SDS

2M Tris-HCl, pH6.8 (BDH)

10% ammonium persulphate (Bio-Rad) Freshly prepared N,N,N’ ,N’-tetramethylethylenediamine (Bio-Rad)

Resolving gels (30 ml) stock solutions

A B C Water TEMED AP 5% 5ml 3.75ml 0.3ml 20.95ml 30pl 300pl 7.5% 7.5ml 3.75ml 0.3ml 18.45ml 30pl 300pl 10% 10ml 3.75ml 0.3ml 15.95ml 30pl 300pl 12.5% 12.5 ml 3.75ml 0.3ml 13.45ml 30pl 300pl 15% 15 ml 3.75ml 0.3ml 10.95ml 30pl 300pl

Stacking gel ( 10ml) stock solutions

A B

c

Chapter 2 Materials and Methods

2 . 6 . 3 Extraction of Triton Soluble Proteins and Measurement of Protein Concentration

Cells were washed twice in ice cold PBS, then extracted for 10 minutes on ice on a rocking platform. The cells were scraped from the tissue culture dishes, and cells debris was

pelleted by centrifugation for 10 minutes at at 4°C. The aliquots of the

suspension (the Triton-soluble portion) were centrifuged as above prior to use. Protein

concentration was measured using the PIERCE BCA protein assay kit. It combines the

reduction of Cu2+ to Cu+ in an alkaline medium (the Biuret reaction) with a

:• - way of measuring the cuprous ion using a reagent containing bicinchoninic acid

(Smith et a i, 1985), which produces a purple colouration which can be measured at

562nm. A standard curve was prepared by making dilutions of a stock BSA solution (2mg/ml, PIERCE). The assay was carried out according to the Manufacturers' instructions. The O.D. at 562 nm was measured for the contents of each well using a Titretek Multiskan MCC/340 MKIl spectrophotometer. The protein concentration of the sample was determined by interpolation from the standard curve.

2 . 6 . 4 SDS-PAGE Electrophoresis

Vertical gel electrophoresis apparatus was used (Atto Corporation). 1.5mm thick gels were prepared between glass plates according to the method of Laemmli (Laemmli, 1970). The composition of the gels is described in preparation of SDS-PAGE gels, above. 1ml of water was applied after pouring the resolving gel to ensure a level, air free interface for polymerisation. Gels were allowed to polymerise for at least 1 hour at room temperature. The water was removed and then the stacking gel was poured. A comb was used to produce wells, and when the gel was set the wells were flushed with the running buffer. (Amersham). Samples and Molecular Weight Marker were added to 10 pi of the loading buffer prior to loading. Samples were boiled at 95-100®C and placed on ice. The samples and Molecular weight markers were applied to the wells and electrophoresed at 60V until the dye just entered the resolving gel. At this time the voltage was raised to 120V until the dye front reached the end of the gel. The gel was then prepared for Western blotting.

2 . 6 . 5 Western Blotting

After electrophoresis, proteins were transferred onto a Millipore Immobilon PVDF (polyvinyldifluoride) membrane which was pre-wetted in absolute methanol using a semi­ dry transfer unit (Biorad) at 200 mA for 45 minutes to 1 hour. After transfer, the filter was stained in Ponceau S solution, to confirm transfer of protein. The membrane was washed

in PBS briefly, and then incubated in 5% milk powder (Premier brands UK limited) in

PB ST (PBS + 0.05% Tween-20) for 1 hour at room temperature or overnight at 4°C to block the non-specific binding of antibody to the filter. Next the filter was washed thoroughly with five changes of PB ST. This was followed by incubation with the secondary antibody conjugated with horseradish peroxidase (HRP - Amersham) in PB ST (no milk powder). After another 5 washes in PB ST, the HRP was detected by a chemiluminescence kit (ECL - Amersham), which was used according to Manufacturers instructions'. If the filter required stripping it was either incubated in the glycine stripping solution for 30 minutes, or incubated in the harsh stripping solution for 30 minutes at 50°C. Regardless of the method of stripping, the filters were subsequently washed in PB ST three times. The filter could then be blocked as normal.