Comprobación de la hipotesis

2.4.1 Western blotting

Although not used extensively in this thesis, SDS polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting presents a useful technique for detection and quantification of proteins. In these studies, SDS-PAGE and Western blotting techniques were employed firstly to determine the conditions under which transfected cDNA encoding the CFP-tagged G a subunits resulted in equivalent protein expression in cell lines, and secondly to show a graded protein expression in response to increasing concentrations o f the antibiotic tetracycline in the inducible cell system (see section 2.2.4).

SDS polyacrylamide gel electrophoresis (SDS-PAGE) is a widely used method for separating proteins according to their molecular weight. SDS is an ionic detergent that denatures proteins by causing unfolding, and then coats the denatured

The polyacylamide gel also contains SDS and is composed o f a matrix created by cross-linkages between two monomers, acrylamide and bisacrylamide, which behaves like a molecular sieve, allowing smaller polypeptides to pass through more readily than larger ones. Most SDS-PAGE is carried out using a discontinuous buffer system in which two layers o f polyacylamide gel are used. The lower layer - known as the resolving gel - is made from polyacrylamide buffered with TrisHCl to pH8.8, and the smaller, upper layer - the stacking gel - is buffered to pH6.8. The whole gel is immersed in a Tris-glycine running buffer containing SDS. The denatured polypeptides are loaded on to the surface of the stacking gel and a voltage is applied (anode at the top, cathode at the bottom). The sample is swept along a steep voltage gradient through the stacking gel and deposited at the surface o f the resolving gel. Here the higher pH of the resolving gel causes ionisation o f glycine, and the polypeptides are no longer swept along but move through the resolving gel in a zone o f uniform voltage and pH. In the resolving gel the migration distance is dependent on the logarithm o f the molecular weight o f the polypeptide.

To visualise the polypeptides resolved on a polyacrylamide gel, the polypeptides must be transferred to another support that allows them to be exposed to antibodies. Nitrocellulose membrane is the most frequently used support due to its high binding capacity for proteins. The wet electrophoretic method of transfer in which the gel is sandwiched with a sheet o f nitrocellulose and subjected to an electrical field was employed in these studies due to its compatibility with the mini-gel electrophoresis system used (Bio-Rad Laboratories, Hemel Hampstead, UK). Following transfer, the nitrocellulose requires treatment with a blocking agent (such as bovine serum

albumin, skimmed milk or Tween) to prevent non-specific binding o f the antibody to the nitrocellulose membrane. Next, the nitrocellulose membrane is treated with the appropriate primary antibody. Then a secondary antibody is applied to detect bound primary antibody. The secondary antibodies used in these studies are conjugated to a dye, horse radish peroxidase (HRP), that allows detection o f labelled bands and transfer o f these bands on to photographic film using a commercially available detection kit (ECL Western blotting detection reagents; Amersham Pharmacia Biotech, Amersham, UK).

Blotting fo r CFP-tagged G-alpha subunits

Cells at 40-50% confluence in 35mm plates were transfected using LIPOFECTAMINE (Gibco BRL, Paisley, UK) with Ipg cDNA for Gia i-CFP, Gja2- CFP, G itts-CFP or GottA-CFP. After six hours the transfection mixture was aspirated fi'om each well and replaced with normal cell culture medium. Cells were then harvested 48 hours post transfection, when confluent, by scraping into ice-cold Tris- buffered saline (50mM Tris, 150mM NaCl, pH7.4; TBS), pelleted in a benchtop centrifuge and resuspended in 250pl o f TBS with protease inhibitors (Complete EDTA-ffee; Boehringer Mannheim, Germany). The protein samples were then denatured by the addition o f 50pl of 6X Gel loading buffer [0.35M TrisHCl (pH6.8), 10.28% (w/v) SDS, 36% (v/v) glycerol, 0.6M dithiothreitol, 0.012% (w/v) bromophenol blue], sonicated for at least 20 seconds and, finally, boiled at 100°C for 5 minutes. The components o f a 12% resolving and stacking gel are listed in the table below. The Acylamideibisacrylamide solution was obtained fi'om BioRad, Hemel

Component: 12% Resolving gel: Stacking gel: 30% solution of 8.0ml 1.3ml Acylamideibisacrylamide 0.5M TrisHCl, pH 6.8 - 2.5ml 1.5M TrisHCl, pH 8.8 5.0ml 10% SDS (w/v) 0.2ml 0.1ml Distilled water 6.8ml 6.1ml Final volume 20.0ml 10.0ml

The gel mixture was allowed to de-gas for 10-20 minutes before the addition of 150jLil 20% (w/v) ammonium persulphate and 20pl TEMED (both from Sigma Aldrich) to the resolving gel. The mixture was mixed by inversion and then poured into the gel apparatus (Mini-gel system, BioRad, Hemel Hampstead, UK). Once the gel had set, polymerisation o f the stacking gel was initiated by addition o f 75 pi 20% ammonium persulphate and 13pi TEMED. The stacking gel was mixed gently and then poured over the resolving gel. A plastic comb was inserted to form wells and the gel was allowed to set for at least 30 minutes before use.

Before the samples were loaded, the plastic comb was removed and the gel apparatus was immersed in a tank containing fresh running buffer (25mM Tris, 250mM glycine, 0.1% SDS, pH8.3). An 80pl volume o f each sample was pipetted into each well. A 5pi volume of pre-stained broad range protein standard markers (BioRad, Hemel Hampstead, UK) was loaded into one well to allow approximation o f the molecular weights o f proteins o f interest, and to serve as a visual marker to indicate whether transfer has occurred on to nitrocellulose. Voltage was then applied across the gel using a power pack (Powerpac 300; BioRad, Hemel Hampstead, UK). The

resolving gel at 80V. For electrophoretic transfer, the gel was removed from the glass plates and placed in a sandwich with nitrocellulose (Hybond ECL; Amersham- Pharmacia Biotech, UK) and filter paper (Whatman n o.l) and enclosed in a plastic cassette. The cassette was then placed in the transfer apparatus and immersed in transfer buffer (48mM Tris, 39mM Glycine, 0.037% (w/v) SDS, 20% (w/v) methanol, pH 8.3). An electrical current o f 250mA was applied across the transfer apparatus for 2 hours at 4°C.

Next, the nitrocellulose membrane was blotted with 5% (w/v) non-fat milk (Marvel, Premier Brands, UK) in PBS + 0.1% Tween (PBS-T) for 1 hour on a shaking platform. This was followed by three washes with PBS-T for a total o f 20 minutes. The primary antibody was then applied to the nitrocellulose membrane in a 5% non­ fat milk (Marvel) solution at a 1:200 dilution. The primary antibody used was a polyclonal rabbit antiserum that recognises GFP and its spectral variants such as CFP (Cat #: 8372-2; Clontech, California, USA). The nitrocellulose was incubated with the primary antibody solution for 12-16 hours on a shaking platform in the cold room at 4°C. After incubation with primary antibody, the nitrocellulose was washed 3 times with PBS-T for a total o f 20 minutes. Then the secondary antibody (Horseradish peroxidase linked anti-rabbit IgG antibody from Amersham Pharmacia, Little Chalfont, UK) was applied to the nitrocellulose at a 1:10 000 dilution in PBS-T and incubated on a shaking platform for 1 hour at room temperature. The nitrocellulose was washed 3 times with PBS-T for a total o f 20 minutes before the reactive bands were visualised using ECL reagents and chemiluminescence film

For both western blots, the aim was to quantify the CFP-tagged protein expression levels. Therefore brief exposures (10-30seconds) were used to develop western blots as the signal can saturate after longer exposures. Furthermore, care was taken to ensure that the total protein content in each sample was similar. For instance, cells were seeded into 35mm wells at identical densities, then transfected (or induced with tetracycline to express the Q a 3-CFP protein in the inducible line) at the same time, and harvested in an identical fashion. Finally, a 20|il aliquot o f each protein sample was kept back and a protein assay was performed (as described in the next section). This confirmed that the amount of protein loaded into each well was virtually identical. For example, for samples induced with tetracycline to express CFP-Gias it was found that the total protein content in each sample was:

[Tet](pg/ml) 0(A ) 0(B ) 0.1 0.5 1.0 10.0

Protein (pg/ml)

16.65 17.03 19.00 18.45 17.38 19.00

2.4.2 Radioligand binding

Radioligand binding techniques were used in this study primarily to quantify the expression levels o f G-protein coupled receptors (GPCRs) expressed stably in receptor + channel stable cell lines. Radioligand binding was performed on crude membrane preparations isolated from the relevant stable lines (HKIR3.1/3.2/A1, HKIR3.1/3.2/Al-Gai.i, HKIR3.1/3.2/a2A, and HKIR3.1/3.2/D2). Large quantities o f these cell lines were grown up (a minimum o f two T175 flasks o f cells were required for a series o f binding experiments). When they had reached confluency, cells were

washed with and harvested into binding buffer (50mM TRIS-HCl, pH 7.4) and stored at -80°C as 1-1.5ml aliquots. They were defrosted slowly on ice and then hypotonically shocked (lOmM TRIS-HCl and lOmM EDTA) on ice for 10 minutes after which an equal volume o f SOOmM sucrose plus lOmM TRIS-HCl was added to restore tonicity. Cells were homogenized using a glass-on-glass Dounce homogeniser. The homogenate was spun at 600g (4°C) for 15 minutes to sediment nuclei and large cell debris. The membrane fraction was obtained by spinning the supernatant at 100,000gav in an ultracentrifuge (Beckman, Optima LE-80K). The pellet was resuspended in binding buffer and incubated with radioligand at room temperature for one hour.

Specific binding was assessed using saturating concentrations o f radiolabelled receptor antagonists: 8nM [^H]-DPCPX for adenosine Ai receptors or the A% receptor-Ga fusion, A i-G iai(C ^G ), 30nM [^H]RX-821002 for adrenergic azA receptors, and 4nM [^H]spiperone for dopamine Dis receptors. Radioligands were obtained from Amersham-Pharmacia Biotech (Little Chalfont, UK). Non-specific binding was determined in the presence o f a 1000-fold excess o f unlabelled antagonist: 8pM DPCPX (A%), 30pM rauwolscine (œia), and 4pM spiperone (Di). Binding reactioms were terminated by the addition o f an equal volume o f ice-cold binding buffer. Bound ligand was separated from non-hound ligand by vacuum filtration onto Whatman GF-B filters, which were then washed 4 times with 2ml ice- cold binding buffer. Each filter was placed in a scintillation vial containing 10ml Ultima Gold M V scintillant (Packard). A Packard TriCarb 2100TR liquid

in triplicate and repeated at least four times. Data were corrected for total protein content in the sample and were expressed as fmol/ pg protein (mean ± s.e.m.).

Protein Assay

Protein concentrations were measured using a standard BioRad protein assay and titrated against a bovine serum albumin (BSA) standard curve. Briefly, 20-50pl o f the crude membrane preparation for each sample was kept back and held at 4°C. A standard curve using known concentrations of bovine serum albumin (BSA) was set up using molecular biology grade BSA (New England Biolabs, Hitchin, UK). The BSA is supplied with restriction enzymes as a lOmg/ml solution, and was used at a 1:100 dilution (0.1 mg/ml). The protein standards were prepared as shown in the table below.

Total protein concentration (pg/ml)

Volume of 1:100 BSA solution (pi)

Volume o f assay buffer (pi) Bio-Rad Protein Assay concentrate 0 0 800 200pl 2 20 780 200pl 4 40 760 200pl 8 80 720 200pl 12 120 680 200pl 16 160 640 200pl 20 200 600 200pl 30 300 500 200pl

Following addition o f 200pl Biorad Protein assay concentrate (Bio-Rad, Munich, Germany), each sample was thoroughly vortexed, incubated for 2 minutes at room temperature and then transferred into a clean plastic cuvette. The absorbance at 595nm was recorded using a spectrophotometer, and this was used to plot the standard curve, g;iving estimates of the protein concentration o f the samples.