F ORMACIÓN ESTÉTICA , ÁREA DE OPORTUNIDAD

The protein content of each of the extracted samples was measured prior to immunoprécipitation and loading on to SDS-polyacrylamide gels. A range of standard dilutions (2000 p.g/ml-50 |ig/ml) of bovine serum albumin (BSA) was prepared using the BCA Protein Assay Kit (Pierce, Rockford, USA), diluted in the same diluent as the sample (i.e. lysis buffer). This kit was used to measure protein concentrations in the samples.

Principle behind the assay

The principle behind the Pierce protein assay is based on the reaction that occurs between copper ions (Cu^*^) in the presence of protein, whereby the copper ions becomes oxidised (Cu^^), and in turn a blue/purple colour is formed. Bicinchoninic acid (BCA) is a selective detection reagent for Cu^^. The presence of protein in combination with Cu^^ (in an alkaline medium) leads to the formation of Cu^^. The purple product, formed by the interaction of two molecules of BCA with one Cu^^ ion exhibits a strong absorbance at 562mn. This allows the spectrophotometric quantitation of protein in aqueous solutions.

Reaction that occurs between the BCA reagents and samples

Protein (peptide bonds) + Cu^^ —> tetradentate-Cu^^ complex

Cu^^ + 2 bicinchoninic acid (BCA) -> BCA-Cu ^ complex (purple coloured)

A mixture of the solutions BCA Reagent A (containing NazCOg, Na4(C0 3)2, bicinchoninic

acid and sodium tartrate in 0.2N NaOH) and BCA Reagent B (4 % cupric sulphate) was prepared (1:50 respectively). 25 pi of each standard or sample was pipetted into separate wells of a 96-microwell plate (GIBCO-BRL, Paisley, UK). 200 pi of solution A/B was added to each well and the plate was agitated on a plate shaker (Luckham RlOO, Luckham Ltd., Sussex, UK) for 30 seconds to combine all the solutions. The plate was covered and incubated at 37°C for 30 min. After incubation the plate was cooled to room temperature and the absorbance at 580 nm of all standards and samples was measured on a plate reader (ICN Flow, High Wycombe, Bucks, UK). A standard curve was plotted for the standards

and the concentration of the protein in each sample was determined by the standard curve. Once the protein concentrations had been determined, 500 |Xg of protein from each sample

was aliquoted into an eppendorf and made up to 300 |xl with lysis buffer. Lysates were stored at -20°C prior to immunoprécipitation.

Immunoprécipitation of protein

Immunoprécipitations were routinely performed by initially preclearing lysates with rabbit IgG and 10 pi protein G-plus agarose beads (Autogen Bioclear, Wiltshire, UK). Lysates were centrifuged at 7 000 g for 5 min at 4°C. The supernatants were transferred to fresh eppendorfs and incubated, rotating, with 3 pg/ml antibody for 2 hrs at 4°C. Antibodies used were raised against focal adhesion proteins: anti-focal adhesion kinase monoclonal antibody (FAK), anti-pl30^“ monoclonal antibody and anti-paxillin monoclonal antibody (all from Affiniti Research Products Ltd, Exeter, UK), anti-FAK polyclonal antibody, recognising the carboxyl terminal and anti-phosphotyrosine antibody PY20 or PY99 (both from Autogen Bioclear, Wiltshire, UK). Immunocomplexes were collected by incubating lysates with protein G Plus-agarose beads for 1 hr-ovemight at 4°C.

After incubation with the agarose beads the eppendorfs were centrifuged for 5 min at 15 000 g. Immunoprecipitates were then washed three times with lysis buffer by centrifugation for 5 min at 15 000 g and the supernatant discarded to leave the agarose:Ab conjugated pellet. Proteins bound to the agarose beads were extracted with 2 x SDS-PAGB sample buffer (200 mM Tris (pH 6.8), 6 % SDS, 2 mM EDTA, 10 % w/v glycerol, 4 % p- mercaptoethanol, 0.01 % bromophenol blue) and boiled for 5 min at 90°C.

Immunoprecipitates were further analysed by SDS polyacrylamide gel electrophoresis followed by Western blotting.

SDS-polyacrylamide gel electrophoresis

The Novex gel apparatus, XCell Mini-Cell (Novex Electrophoresis GmbH, Frankfurt, Germany) was used for all polyacrylamide gel electrophoresis. For most proteins Mw ranging from 100 to 200 kDa (e.g. FAK and p i30^^) 7.5 % acrylamide separating gels were overlaid with a 4 % acrylamide stacking gel. For proteins with a Mw 40-100kDa (e.g. paxillin) 10 % acrylamide separating gels were used. The separating gel (50 %

Acrylamide/Bis, 1 M Tris-HCl (pH 8.8), 10 % SDS, 50 % sucrose, 75 |xM ammonium persulphate, TEMED and water) was routinely poured first into the gel cassette and overlaid with t-amyl alcohol (Sigma, Poole, UK) and allowed to polymerize for at least 1 hr. The t-amyl alcohol was removed and the gel surface washed extensively with ddH20. The stacking gel (50 % Acrylamide/Bis, 0.375 M Tris-HCl (pH 6.8), 10 % SDS, TEMED, ammonium persulphate and water) was poured on top of the separating gel and the combs were positioned at the top of the stacking gel immediately after pouring. The stacking gel was allowed to polymerise for 1 hr. The gel apparatus was assembled and running buffer (24 mM Tris, glycine, 10 % SDS, ddHzO) was poured into the gel tank. Samples to be electrophoresed were boiled for 5 min prior to loading onto the gel. The gels were routinely run at 125 v -130 v for 2 hrs. For each gel, 3 |il of a molecular weight marker was also loaded (“See Blue” marker, Novex Electrophoresis GmbH, Frankfurt, Germany).

Western blotting

Once the samples had reached 1 cm from the bottom of the gel, the gel was removed from the gel cassette and the separated proteins were transferred from the gel to Hybond^^ ECL^^ nitrocellulose membrane (Amersham Life Science, Bucks, UK) using the Novex XCell Mini-Cell. The cassette was assembled with the gel sponged between 3MM paper (Whatman, UK) and the nitrocellulose membrane. The nitrocellulose membranes and the 3MM paper were pre-wetted in transfer buffer (12 mM Tris, 96 mM glycine, ddHzO, pH 8.3) for 10 s. The membrane was laid on top of the gel on the side nearest the positive electrode. Any air bubbles formed between the gel and the membrane during assembly of the cassette were rolled out (Figure 2.4). The proteins were transferred at 25 v for 2 hrs in transfer buffer. After transfer, the cassette was dismantled and the membrane was stained with Ponceau Red (Sigma Chemicals, Poole, UK) to ensure complete and even transfer of all proteins. The membrane was rinsed with ddHzO to remove excess Ponceau Red dye. The membranes were blocked in 5 % w/v non-fat milk protein (Marvel) in TBS (pH 7.6) containing 1.0 % w/v Tween 20 (Sigma Chemicals, Poole, UK) (TBS-T) or for phosphotyrosine antibodies, 1 % w/v BSA with 1 % milk w/v in TBS-T for 1 hr at room temperature or overnight at 4°C. After incubation, the blocking solution was discarded and the membranes were rinsed twice in TBS-T then incubated with protein-specific antibodies (anti-phosphotyrosine antibody, PY20 or PY99, Autogen Bioclear, Wiltshire, UK) or focal adhesion protein antibodies in 5 % w/v Marvel dissolved in TBS-T or 1 % w/v BSA / 1 % w/v Marvel for 1 hr at room temperature or overnight at 4°C. This was discarded and the membranes were washed 3 x 5 min in TBS-T. The membranes were then incubated with horseradish peroxidase (HRP) conjugated anti-mouse IgG (Sigma Chemicals, Poole, UK) at

+

nitrocellulose

gel 3 M M

paper sponges

Figure 2.4. Assembly of gel cassette for transfer of proteins from the gel to nitrocellulose membrane.

a 1:5000 dilution in 5 % w/v Marvel dissolved in TBS-T for 1 hr at room temperature. Finally the membranes were washed in the same way as described above. Immunoreactive bands were visualised by incubating the membrane in ECL (Amersham Life Science, Bucks, UK) reagent for 1 min and then exposing the membrane to autoradiography film (Hyperfilm^'^, ECL''’''^-high performance chemiluminescence film, Amersham Life Science, Bucks, UK) for 30 s and 1 min.

Films were immediately developed using a Velopex MD2000 developer (Medivance Ltd., UK). ECL western blotting is a light emitting non-radioactive method for detection of immobilised specific antigens conjugated with HRP-labelled antibodies.

Principles of ECL detection

Chemiluminescence involves the excitation of chemicals effected by a chemical reaction. HR? (to which the secondary antibody is conjugated) oxidises luminol (present in the ECL reagents) in the presence of chemical enhancers e.g. phenol. Immediately following oxidation, luminol is in an excited state. Luminol decays to ground state via a fight emitting pathway. The presence of phenol on the reaction has the effect of increasing the fight output approximately 1000 fold and extending the time of fight emission. The fight produced by luminol returning to ground state peaks after 5 - 2 0 min and decays slowly thereafter with a half fife of 60 min. The maximum fight emission is at a wavelength of 428 nm which can be detected by a short exposure to blue-light sensitive autoradiography film.

2.7 IMMUNOFLUORESCENT STAINING OF FOCAL ADHESION PROTEINS