La concepción estructuralista de las teorías

A Western blot method is commonly used to detect the presence of a determined protein in cell culture or tissues. The technique consists in two main steps: the first is the polyacrylamide gel electrophoresis (PAGE) where the proteins present in a solution are separated on the basis of their molecular weight; the second process, the immunoblot, is the transfer of the separated proteins from the polyacrylamide gel to a membrane that is then incubated with an antibody against the protein of interest and then detected with an enzyme- labelled antibody directed against the primary antibody.

In my thesis I have used the Western blot technique not to detect the presence of a certain protein in a solution, but to assay sera from treated and untreated animals for the presence of antibodies against adenoviral proteins. For this reason, cell lines were infected with luciferase or LacZ-encoding adenoviruses or left uninfected and the cell lysates obtained underwent electrophoresis and immunoblot. At this stage, blotted membranes were incubated with sera from AdV- or control-treated animals culled at different time-points that were used as primary antibodies. As a principle, instead of using a primary antibody with known specificity to detect the presence of the specific target protein on the blotted membrane, membranes containing adenoviral proteins were used to detect the presence of specific antibodies in the sera. Enzyme-conjugated anti- mouse antibodies were used in the last step to detect anti-viral antibodies bound to the viral proteins.

To achieve this, confluent Hek293 cells were infected with LacZAdV, LucAdV or left uninfected. Cells were returned in the incubator at 37˚C for 48 hours. At the

end of the incubation period, the medium was discarded and the cells washed in PBS. A lysis buffer (RIPA buffer, 50mM Tris, 150mM NaCl, 0.5% sodium deoxycholate, 1% NP-40, 0.1% sodium dodecyl sulphate, pH=8) containing protease inhibitors (Complete mini EDTA-free, Roche) was then added to the wells to disrupt plasma membranes and free and stabilise the host and viral proteins.

The protein content of the lysates was quantified with a bicinchoninic acid (BCA) assay (Pierce, USA). Cell lysates were diluted in a 5x Laemli solution (10% sodium dodecyl sulphate, 50% glycerol, 25% β-mercaptoethanol, 0.01% bromphenol blue and 0.312 M Tris HCl, pH=6.8) and left on a hotplate at 100˚C to denaturise for 5 minutes. β-mercaptoethanol is a reducing agent, which helps breaking the sulphide bonds among peptides. The sodium dodecyl sulphate (SDS) molecules present in the solution and in the lysis buffer help to denaturise the proteins as the non-polar moiety binds to the hydrophobic amino acids of the proteins exposing the negative portion of SDS to the buffer and giving thus a negative charge to the hydrophobic residues of the protein. Moreover, the negatively charged moiety of SDS also binds to the positive residues, neutralising them. Eventually, during the treatment at 100ºC, disulphide bonds are reduced by β-mercaptoethanol and the high temperature allows denaturation of the proteins into their primary structure that is maintained by SDS molecules by neutralising the positive charges and giving to the polypeptide a strongly negative charge. Bromophenol blue is a blue dye that helps monitoring the migration of the proteins in the next steps.

The cells lysates were loaded on a commercial polyacrylamide gel (Nu-PAGE, 4-12% bis-tris, Invitrogen) in an electrophoresis tank (Invitrogen) filled with

running buffer (5mM MOPS, 5mM Tris, 0.1% SDS, 0.1mM EDTA) and a constant electric tension of 100V was applied to the tank for approximately 1.5 hours. The proteins suspended and denatured in the loading buffer have a strong negative charge and when the electric field is applied, they migrate into the polyacrylamide towards the anode. During the migration the proteins separate in bands according to their size and the composition of the gel matrix: smaller peptides migrate faster, while bigger proteins migrate slower. Per every gel lane I loaded the equivalent of 25 µg of protein. At the end of the electrophoresis, the separated bands were transferred to a nitrocellulose membrane with the aid of a commercial semi-dry system (iBlot, Invitrogen) according to manufacturer’s instructions. The efficiency of the transfer was assessed incubating for few minutes the membrane with a 0.1% Ponceau red solution in acetic acid: Ponceau red binds proteins in a non-specific manner and thus allows a rapid visual check of a successful and equal transfer. The membrane was then washed from the red dye for 10 minutes in TBST (TBS 0.1% Tween-20) on a rocking shaker. The membrane was then incubated in a solution containing 5% powder milk in TBST for 3 hours at room temperature. Incubation with the milk solution blocks protein non-specific binding.

At this point, membranes were incubated either with a commercial anti- adenovirus antibody (goat anti-adenovirus 5 whole antiserum, AB6982, Abcam, dilution 1:4’000) as a positive control or with sera from the treated mice at a dilution of 1:100 in a 5% milk TBST solution, overnight at 4˚C. The membrane was then washed 3 times for 5 minutes in TBST and incubated with a horseradish peroxidase-conjugated detection antibody against the primary (in this case goat anti-mouse IgG from Sigma for the membranes incubated with

mice sera or rabbit anti-goat IgG from Dako for the membranes incubated with the commercial anti-adenovirus antiserum). Secondary antibodies were diluted in blocking solution and the membranes incubated for 1 hour at room temperature.

To detect the binding of the detection antibody to the protein of interest, an enhanced chemiluminescence (ECL) substrate is used. The ECL substrate contains luminol that is oxidised by the peroxidase-linked antibodies present on the membrane. Luminol in its excited state decays to the ground state emitting light at 428 nm. The light emitted from the membrane is used to expose an X- ray film that is then developed. After 3 washes in TBST the membrane was incubate with ECL (Amersham Life Science), wrapped in transparent film and left at room temperature for one minute. The membrane was then used to expose an autoradiography film for various times in a dark room equipped with red-light illumination. After exposure the films were developed in an automated developing machine and studied.