La Autonomía de la Voluntad

2.1

Materials

2.1.1 Chemicals and media

Unless an alternative source is indicated in parenthesis when first mentioned, all chemicals and reagents described herein were purchased from Sigma, with the exception that acetic acid, dimethyl sulphoxide (DMSO), ethanol, glycerol, hydrogen peroxide solution, methanol and sodium dodecyl sulphate (SDS) were purchased from Merck. Distilled water, Luria-Bertani broth, phosphate buffered saline (PBS), tissue culture media and trypsin/versene solution were prepared by the Imperial Cancer Research Fund (ICRF) Research Services Department.

2.1.2 Antibodies

Antibodies used in this study were from the following sources: monoclonal anti- phosphotyrosine clone PY72, monoclonal anti-ErbB l clone F4 (ICRF Research Monoclonal Antibody Department), rabbit polyclonal anti-mouse epidermal growth factor (Sigma), rabbit polyclonal anti-human erythrocyte catalase (Calbiochem), Cy5- labelled goat anti-rabbit IgG and Cy5-labelled donkey anti-mouse IgG secondary antibodies (Jackson Immunoresearch Laboratories). Cy3-labelled goat anti-rabbit IgG secondary antibodies were generated by labelling purified IgG (Sigma) with Cy3 as described (see section 2.2.3).

2 .\3 Plasmid DNA

Plasmid DNA was amplified in E.coli using standard molecular biology techniques (Sambrook et al., 1989) and purified using a commercially available kit (Midi Prep kit, Qiagen). Plasmid DNA constructs used in the work for this thesis are listed in Table 2.1.

Table 2.1 Plasmid DNA constructs

Construct Backbone vector Source

ErbBl-GFP pEGFP-C3 (Clontech) Dr. F. Wouters

Cell Biophysics Laboratory ErbB3-GFP pEGFP-C3 (Clontech) Dr. F. Wouters

Cell Biophysics Laboratory ErbBl pCDNA3 (Invitrogen) Dr. C. Dickson,

Viral Carcinogenesis Laboratory, ICRF

Human fibroblast catalase PZeoSV2 (Invitrogen) Dr. J. Mendelez,

Albany Medical College, New York

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2.1.4 Formulation of commonly used reagents

4% paraformaldehyde. 8 grams of paraform aldehyde (PFA) pow der was

dissolved in 150 ml of distilled water (supplemented with 1 ml of concentrated sodium hydroxide solution) with stirring at 50°C. After the solution had cleared, 20 ml of lOX PBS was added and the pH of the solution was brought to 7.4 with hydrochloric acid. Finally, the solution was brought to a volume of 200 ml with distilled water and filtered through a vacuum-driven 0.22 p,m filter flask (Nalgene). The 4% PFA solution was stored at 4°C for a few weeks or for months at -20°C. Just prior to use the PFA solution was filtered once more through a M illex-GP 0.22 pim syringe driven filter unit (Millipore).

Mowiol mounting solution. 2.4 g of Mowiol 4-88 (Calbiochem) was mixed with 6

ml glycerol and 6 ml distilled water. The mixture was vortexed and then shaken for 2

hours. 12 ml of 200 mM Tris pH 8.5 was added and the mixture was incubated at 50°C with stirring until the Mowiol had dissolved (typically 24-48 hours). The solution was then filtered through a Millex-GP 0.45 p,m syringe driven filter unit (Millipore) and stored in aliquots at -20°C. When aliquots were defrosted they were kept for a few weeks at 4°C for practical use. The solution was allowed to warm to room temperature before use.

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2.2

Methods I : Protein chemistry

2.2.1 Biorad protein assay

18 protein concentration standards in the range 2.4-24 pg/ml were made by diluting a 200 pig/ml bovine serum albumin solution in distilled water. 150 pi of each protein standard solution was pipetted in duplicate into the wells of a 96-well plate. Solution A was generated by mixing 1. 6 ml of protein assay reagent (Biorad) with 4.4 ml

of distilled water. 50 pi of solution A was added to each well of the 96-well plate containing the protein standards or containing 150 pi of the protein sample, which was diluted in duplicate at various concentrations within a similar range to the protein standards. The absorbtion of each well was measured at 595 nm in a 96-well plate reader (Biorad) and protein concentrations were calculated using the plate reader software (Biorad).

2.2.2 Generation and purification of antibody Fab fragments

Purified monoclonal PY72 antibody was obtained in sterile filtered PBS from the ICRF Research Monoclonal Antibody Department. The antibody was concentrated to a protein concentration of 15-20 mg/ml and the buffer exchanged for 20 mM Na- phosphate, 10 mM EDTA, pH 7.0. This was performed using a disposable YMIOO Centricon centrifugation concentrating device (100 kDa molecular weight cut-off; Amicon). The digestion reaction consisted of 250 pi of the concentrated antibody preparation (-- 4-5 mg), 500 pi of digestion buffer (20 mM Na-phosphate, 10 mM EDTA, 20 mM cysteine / HCl , pH 7.0) and 500 pi of a 50% slurry (in digestion buffer) of papain immobilised to agarose beads. Digestion was performed for 6 hours at 37°C with

vigorous shaking (400 RPM on a rotating platform).

The Fab fragments were separated from Fc fragments and non-digested antibodies using a protein A-sepharose column (Econo-Pac 2 ml column, BioRad). The column was equilibrated with 20 bed volumes (40 ml) of 20 mM Na-phosphate buffer pH 7.0, before applying the digestion reaction (1250 pi) to the upper bed of the column. After the digested material had moved into the column matrix a further 2 ml of equilibration buffer

________________________________________________________________ Chapter! was added. The flow-through, which contains purified Fab fragments, was collected in 25 X ~200 p.1 fractions. To identify which fractions contained the purified Fab fragments, the absorbance was measured at 280 nm in a UV-visible spectrophotometer (Agilent technologies) and the peak fractions were pooled. The pooled fractions were then concentrated to a volume below 0.5 ml and buffer exchanged into PBS using a YMIO Centricon centrifugation concentrating device (10 kDa molecular weight cut-off; Amicon). Fab fragments were fluorescently labeled as described in section 2.2.3.

2 2 3 Labelling antibodies with Cy dyes

The monofunctional Cy dyes used here are indocyanine fluorophores that are derivatised with a single N-succinimidyl ester group to facilitate covalent coupling to primary amino groups and were obtained from Amerhsam Pharmacia Biotech. The ester group may be hydrolysed by water which destroys the reactivity of the dye with amino groups. To prevent the dye from reacting with condensed water, a single aliquot of lyophilised Cy dye was removed from 4°C storage just prior to labelling and allowed to warm to room temperature before opening. The solid was dissolved in 20 pil of dry NJM- dimethylformamide (DMF). The precise concentration of dye present in solution was determined by diluting the dye solution 1:10000-1:40000 in distilled water and measuring the absorbance in a 1 cm light-path cuvette. The concentration of the dye (M) was then calculated from the following formula: