2.5.1 Preparation of protein samples for use in assays
2ndorder branches of the pulmonary artery were identified as described in section 2.1 and dissected clear of surrounding parenchyma. Arteries were then cut open longitudinally and the endothelium was removed by gently rubbing of the luminal wall with a cotton bud. The brain of the animal was removed from the skull and cut into pieces, as was the heart and skeletal muscle (the vastus lateralis from the hind leg). All tissue was snap frozen immediately after removal in liquid nitrogen and stored at -80 oC until required. Tissue was placed into a small cooled mortar containing liquid nitrogen, once most of the liquid nitrogen had evaporated a cooled pestle was used to grind the tissue into a fine powder. The crushed tissue was then placed in a cooled microcentrifuge tube and buffer was added (3l per 1 mg tissue) of the following composition (mM): 50 Tris base, 150 NaCl, 50 NaF, 5 Na pyrophosphate, 1 EDTA, 1 EGTA, 1 DTT, 0.1 benzamidine, 0.1 % (v/v) Triton X-100, 0.1 PMSF, 0.25 mannitol, pH 7.4. After two minutes a motorised pestle was used to ensure a smooth homogenate was formed. Once formed the homogenate was left on ice for 30 minutes after which time the homogenate was spun on a desktop centrifuge for 5 minutes at 10,000 g, this was carried out at 4oC. The supernatant containing the protein fraction was removed to a cooled eppendorf tube and both the supernatant and the pellet containing cell debris were retained at -80oC until required.
2.5.2 Bradford assay of protein content
Since it was first described in 1976, the Bradford assay has become a widely used protein assay method as it is simple, rapid, inexpensive and sensitive (Bradford, 1976). The Bradford assay takes advantage of the absorbance shift of Coomassie brilliant blue G-250 (CBBG) when it binds to proteins at specific amino acid residues, namely arganine, tryptophan, tyrosine, histadine and phenylalanine residues. CBBG binds to these residues in its anionic form which has an absorbance maximum of 595 nm. The free dye in solution is found in the cationic form, with an absorbance maximum at 470 nm. The assay is read at 595 nm in the spectrophotometer which gives a measure of the CBBG complex with the protein.
Appropriate volumes (1-10 l) of each protein sample were added to different plastic cuvettes. To each cuvette 2 ml of Bradford reagent was added, cuvettes were then shaken to mix contents. Bradford reagent consists of Coomassie brilliant blue G-250, 95 % ethanol and 85 % orthophosphoric acid. The spectrophotometer (CE 393 Digital Grating Spectrophotometer, Cecil Instruments Ltd., UK) was turned on and allowed to warm up for fifteen minutes in order to allow for a consistent reading. Protein concentrations are determined by extrapolation from a standard curve of protein concentrations. This was produced using a BSA stock solution (1.45 mg/ml) diluted to the following concentrations, in pure-filtered H2O (mg protein/ml): 0, 0.1, 0.25,
0.5, 0.75, 1, and 1.45. The standards and the protein samples were both read on the spectrophotometer and the protein concentrations of the tissue samples were determined via interpolation using Prism software described in section 2.6.6.
2.5.3 SDS-polyacrylamide gel electrophoresis
SDS- polyacrylamide gel electrophoresis (SDS- PAGE) is a method for separating and identifying proteins according to their molecular weight. The proteins move through the gel due to the influence of an electric current and the proteins migrate towards the anode, the positive electrode.
The pores within the gel restrict the passage of the proteins in proportion to their molecular weight so that the low molecular weight proteins move more rapidly. In this system gels of two different pore sizes are used: the stacking gel has a lower pH (6.8) and a larger pore size than the resolving gel (pH 8.8). This results in the concentration of the proteins at the interface between the two gels, thus giving a better resolution. Effectively, the gels are made by chemical polymerisation of a mixture of acrylamide and bis- acrylamide (a cross linker), with ammonium persulphate which catalyses and TEMED which initiates the polymerisation reaction. The final concentration of acrylamide depends on the molecular weight range desired, high concentration gives good resolution of low molecular weight proteins and vice versa. Resolution of proteins was performed using 6 % bis- acrylamide gels cast in Novex gel cassettes (Invitrogen, UK) with five lanes. The resolving gel mix (6 % acrylamide, 0.375 M Tris, 0.1 % w/v SDS, 0.05 % w/v APS and 0.05 % TEMED) was poured into the gel cassette. The mix was overlaid with pure- filtered H2O and left to polymerise for 30 minutes. Once the gel had
polymerised, removal of any unpolymerised gel mix was achieved by washing with distilled H2O. After this the stacking gel (4.2 % acrylamide, 1 % w/v
SDS/ 0.125 M Tris, 0.05 % w/v APS and 0.08 % TEMED) was poured over the resolving gel and a 10 well comb was inserted to create lanes. The gel was then allowed to polymerise again for approximately 30 minutes.
Proteins are boiled in Laemmli buffer containing SDS, a reducing agent (-mercapotethanol), glycerol and the marking dye bromophenol blue. Sodium dodecyl- sulphate (SDS) is a detergent with a strong negative charge, which binds avidly to all proteins, hence all proteins, whatever their original charge, are converted to complexes that have a strong negative charge. Also, the three dimensional shape of the protein is converted into a rod-like structure. Since the SDS molecules bind to polypeptides with a constant weight ratio, the charge per unit weight is constant and electrophoretic motility becomes a function of molecular weight. The reducing agent breaks the disulphide bonds (-S-S-) within the proteins which aids in the conversion of the proteins 3- dimensional structure into a rod- like structure and prevents it from converting back. The presence of the marking dye allows control over the distance of
migration of proteins. Using the protein concentrations determined from the Bradford assay, protein samples were made up to a final concentration of 1 mg/ ml in the appropriate volumes of sample buffer (composition (mM): 63 Tris
pH6.8, 139 SDS, 3.3 % glycerol, 1 % bromophenol blue, 5 % -
mercaptoethanol) and 50 mM Tris. The resultant mixture was heated to 95 oC for five minutes after which 50g of sample was loaded onto the gel. The gels were then run using an XCell surelock Mini-Cell system (Invitrogen, UK). In order to allow size discrimination of the resolved proteins, 5l of Broad Range Prestained protein markers (Bio-Rad, UK) were also loaded into one lane. The gels were run for 0.5 hours at 50 V to allow protein to concentrate at the gel interface. Following this, gels were then run at 125 V for 2.5 hours.
2.5.4 Immunoblotting
Following SDS- PAGE the gels were removed from the cassettes and placed into transfer buffer of the following composition (mM): 42.9 Tris, 38.9 glycine, 0.038 % w/v SDS, 20 % methanol. Grade 1F electrode filter paper and Hybond ECL nitrocellulose membranes were cut to the size of the gel and pre- soaked in the transfer buffer. Proteins were transferred onto nitrocellulose membranes using an XCell II blot module Mini Cell system (Invitrogen, UK). A stack was built in order to transfer the proteins. On top of a piece of filter paper was placed the gel. The ‘sticky’ nitrocellulose membrane was placed onto the gel and a further piece of filter paper was placed on top of this. Trapped air was removed from the stack by gently rolling a glass rod across the top. Two blotting pads soaked in transfer buffer were placed on the cathode and the stack was placed on top of this. A further 2-3 blotting pads were placed on top of the stack and the anode was placed on top of the stack. Following this the gels were blotted for 1.5 hours at 25 V (~ 100mA).
2.5.5 Immunodetection
After transfer, the nitrocellulose membranes were blocked with 5 % blocking buffer of the following composition (mM): 20 Tris base, 150 NaCl,
0.1 % Tween 20, 5 % non- fat milk powder, pH 7.5) for 1 hour at room temperature. The blocking buffer acts to bind to the remaining sticky areas of the membrane in order to prevent any non- specific protein interactions between the membrane and the antibody protein. Following this, the membranes were washed in TBST of the following composition (mM): 20 Tris base, 150 NaCl, 0.1 % Tween 20, pH 7.5) three times for 10 minutes. The blots were incubated with the primary antibody diluted in 1 % Blocking buffer of the following composition (mM): 20 Tris base, 150 NaCl, 0.1 % Tween 20, 1 % non- fat powder milk, pH 7.5) overnight at 4oC.
The following day the membranes were washed 2 X 5 minutes and 1 X 15 minutes with TBST to remove any unbound primary antibody. Secondary HRP- conjugated anti-rabbit antibodies diluted in 1% blocking buffer at 1:1000 were incubated with the membranes for 2 hours. The secondary antibody is linked to horseradish peroxidase (HRP), which can be used to allow visualization of the protein of interest on the membrane. The membranes were then washed for 2X 5 minutes and 1 X 15 minutes in TBST. The binding of the primary antibody was detected using a chemiluminescence detection system. The membrane was incubated with the substrate for the HRP, luminol, which is a diacylhydrazide. This reaction was carried out in the presence of chemical enhancers such as phenols which increase the light output and extend the time of light emission. The HRP catalyses the oxidation of luminol which excites an electron, moving it to a higher energy state. This electron then decays back to its resting ground state which emits a photon of light. The maximum emission is at the wavelength 428 nm that can be detected by a short exposure to blue- light sensitive autoradiography film. The chemiluminescence system used was the ECL Western blotting detection reagents (Amersham Bioscince, UK). The membranes were incubated with a 1:1 mixture of Reagents 1 and 2 for 1 minute. Following this, the membranes were exposed to ECL Hyperfilm (Amersham, UK) in a dark room. Exposure times ranged from 1 minute to 5 minutes depending on the strength of the signal. The X-ray films were developed using a Fuji film X-ray processor (Model RGII).
2.6 Fluorescence imaging of Ca2+ within isolated pulmonary artery