Aptitud al servicio

Background: Western blotting is a technique that allows the visualisation of specific proteins following SDS-PAGE. The proteins are transferred from the acrylamide gel to either polyvinylidene fluoride (PVDF) or nitrocellulose membrane. The transfer takes advantage of the negative charge imparted on the proteins by SDS. The membrane must be first activated, the gel and membrane are then held tightly together and an electric current causes the proteins to move onto the membrane. The activated membrane has a high affinity for proteins, so to prevent any further proteins binding and leading to non-specific binding (from the antibody in later steps) it must first be blocked with milk. The membrane is then incubated with an antibody against the protein of interest. The excess is washed away and the membrane is then incubated with a secondary antibody against the primary antibody. This secondary antibody is conjugated to the horseradish peroxidase (HRP) enzyme, and enhanced chemiluminescence (ECL) can then allow visualisation of the protein.

HRP can covert luminol to its oxidised form in the presence of hydrogen

peroxide, this reaction produces light as a by-product, which can be detected on x-ray film in an imaging hood. The amount of protein present in the band will be directly relative to the amount of light observed. Meaning western blotting is a semi-quantitative method for a single band but cannot be directly compared to separate blots or different protein bands on the same blot.

42 Reagents:

 Transfer Buffer: 39mM glycine, 48mM Tris-base, 0.0375 % (w/v) SDS and 20 % (v/v) methanol

 Tris-buffered saline-Tween (TBS-T): 10mM Tris-HCl pH 7.4, 0.15M NaCl and 0.2 % (v/v) Tween-20

 Non-fat dry milk (Marvel brand)

 HRP-conjugated secondary antibody (Dako, Agilent Technologies, Santa Clara, USA)

 ECL reagents

 Bovine serum albumin (BSA)

 PVDF or nitrocellulose membrane

 Methanol

Method: Transfer was performed using a semi-dry method throughout this project. 4 pieces of blotting paper were cut to the size of the gel being

transferred; these were then placed into transfer buffer. The membrane was cut marginally smaller and activated by being placed in methanol, then moved to transfer buffer. A sandwich was made with the components: in order, two pieces of blotting paper, the membrane, the gel and then two more pieces of blotting paper. Any bubbles in the sandwich were removed with a roller. It was then secured and moved to electroblotter from BioRad (Hercules, USA).

Electroblotting was undertaken at 80 mA per gel for 1.5 hours. The membrane was then washed three times in TBS-T before being left in 5% milk for 2 hours at room temperature. After which, the membrane was again washed three times in TBS-T for 5 minutes, before being left overnight at 4°C in primary antibody (table 2.2). The primary antibody was diluted to manufacturer-recommended concentration in 5% BSA in TBS-T with 0.02% sodium azide. The next morning the membrane was washed three times in TBS-T for 5 minutes. The relevant secondary antibody was then added at 1:2000 concentration, in 5% milk. The secondary antibody was left for 1 hour, then followed by three final wash steps with TBS-T for 5 minutes. The membranes were treated with the ECL

reagents and visualised using a Gbox EF Gel Documentation System with Genesnap software (Syngene, Cambridge, UK).

43

2.1.13 Small interfering RNA (siRNA) transfection Background:

siRNA transfection allows the short-term silencing of a specific gene of interest.

It takes advantages of a process that is normally important in protecting the cell against viral infection. The silencing is mediated by double stranded (ds)RNA which is delivered intracellularly using lipid vesicles that can permeate the cell membrane. Once in the cell the dsRNA is cleaved by the enzyme

endoribonuclease Dicer which leaves 21-23bp siRNAs. These in turn bind to the RNA-induced silencing complex (RISC). This is a ribonucleoprotein which

incorporates the siRNA and uses it as a template to target mRNA. Once the target mRNA is found a protein in the RISC, called Argonaute, then activates and cleaves the mRNA. This can essentially silence the specific gene in a given cell population. After transfection, cells can be either harvested to test

transfection efficiency, or treated further to test the effect gene silencing will have on the cells response to stimuli.

Reagents:

 Dharmacon ON-TARGET plus SMARTpools (Thermo Fisher, Loughborough, UK).

Table 2.2 Antibodies used throughout this project.

44

 Dharmacon ON-TARGET plus Non-targeting pool control siRNA (Thermo Fisher, Loughborough, UK).

 DharmaFECT Transfection reagent (Thermo Fisher, Loughborough, UK).

 5x siRNA resuspension buffer (Thermo Fisher, Loughborough, UK).

 Serum-containing medium: DMEM-F12, 10 % (w/v) FBS, 2mM L-glutamine, 200IU/ml penicillin, 200µg/ml streptomycin.

 Antibiotic- and serum-free medium: DMEM-F12, 2mM L-glutamine.

 Serum-free medium: DMEM-F12, 2mM L-glutamine, 200IU/ml penicillin, 200µg/ml streptomycin.

 PBS

 RNase-free water

Method: SW1353 cells were cultures in 96 or 6 well plates until they had

reached 50-70% confluency. siRNA including the non-targeting siRNA controls were made up at a final concentration of 100nM, from a 20µM stock, using 1x siRNA resuspension buffer, diluted in RNase-free water. DharmaFECT

transfection reagent (0.2µl/well of 96-well plate or 3.2µl/well of 6-well plate) was added to antibiotic- and serum-free medium (10µl/well of a 96-well plate or 160µl/well of a 6-well plate). In a separate container the volume of siRNA

required for the desired transfection concentration was made up to 10µl/well (96 well) or 160µl/well (6 well) with Anti-biotic and Serum-free medium. Both

DharmaFECT and siRNA were left for 5 minutes, then combined and left for a further 20 minutes all at room temperature but under sterile conditions. 90µl/well (96 well) or 1440µl/well (6 well) of serum-containing medium was then added.

Cells were washed and then 100µl/well (96 well) or 1600µl/well (6 well) of the final mixture was added to the cells and left for 24 hours. After which, they were washed with PBS, serum starved overnight and then subsequently harvested or further treated.