blotting
4.2.2.1.6.1. Preparation of samples for analysis by SDS PAGE
Following the appropriate stimulation, the 6 well plates were removed from the incubator at 370C and placed on ice. Media was aspirated from each plate, and the cells were washed twice with 500 µl ice-cold HPFEV buffer (50 mM Hepes, 10 mM sodium pyrophosphate Na4P2O7, 100 µM sodium fluoride NaF, 4 mM EDTA and 2 mM orthovanadate Na3Vo4, pH7.4) to stop the reaction and inhibit protein dephosphorylation. Following buffer aspiration, adherent cells were harvested by scraping into 400 µl hot (650C) Laemmli sample buffer (0.048 M Tris-HCL, pH 6.8 containing 0.8 mM sodium pyrophosphate, 5 mM EDTA, 2% w/v SDS, 10% glycerol, 001% w/v bromophenol blue, 50 mM DTT) (Laemmli, 1970) and pulling the sample passed repeatedly through a 23G needle. The cells were then heated at 1000C for 5 min in a heating block, in order to denature the protein present, and then stored at -200C until analysis.
4.2.2.1.6.2. Determination of protein concentration
The amount of protein in cell lysates was quantified to ensure equal loading in Western blot gels. Total protein in each HUVEC sample (without bromophenol blue) was analysed with Bradford protein assay kit (Bio-rad 500-0120) according to the manufacturer’s instructions, with the detergent
142
compatible adaptation and the use of bovine serum albumin as standard (200, 400, 600, 800, and 1000 mg/ml). Initially, dilutions of the protein standard were prepared at above concentrations then 5 µl of the standard or cell lysate were added to individual wells of a 96 well-plate in triplicate. 25 µl of reagent A* (2% v/v reagent S in reagent A) was added to each well and then and then 200 µl of reagent B were added onto each well. The plates were left for 15 min at room temperature with gentle agitation to mix the reagents. The absorbance was read by 96 well plate reader at 750 nm, each sample producing a triplicate reading to allow the average of these triplicates to be calculated. The volume from each sample containing 30-40 µg of protein was then calculated and the appropriate volume of sample used for electrophoresis.
4.2.2.1.6.3. Resolution of protein by SDS-PAGE
A Bio-Rad mini protein gel electrophoresis kit (500-0120) was used to run all gels. Tris buffers were prepared and stored at room temperature for both resolving gel (1.5 M Tris base and 0.4% w/v SDS, pH 8.4) and stacking gel (0.5 M Tris base and 0.4% w/v SDS, pH 6.8). 10% resolving gel was prepared and poured in between glass plates and spacers (1.0 mm thick) to a level 1cm below the bottom of the comb when inserted and overlaid with 0.1% w/v SDS solution. Once the resolving gel polymerized, SDS was removed and a 3% stacking gel was poured on the top and a 1mm comb was inserted. Following gel polymerization, they were assembled and placed in a Prespex tank containing electrophoresis buffer (25 mM Tris base, 192 mM glycine and 0.1% w/v SDS). Appropriate volume from each sample containing 30-40 µg protein was loaded per well as well as 5 µl prestained
143
protein ladders (Thermo Fisher Scientific, UK) and biotinylated standards (New England Biolabs, UK). Samples were electrophoresed at a constant blotting buffer (25 mM Tris base, 192 mM glycine and 20% v/v methanol) for 5 min each. A transfer sandwich was prepared containing black cassette, sponge, filter paper, resolving gel; pre wet PVDF membrane, filter paper and white cassette. This sandwich was inserted into a transfer tank buffer filled with blotting buffer. The whole tank was engulfed with ice and connected to the power supply at 100V for 2.30hr.
4.2.2.1.6.5. Detection of phospho-p38 MAPK, total p38 MAPK proteins, Bcl-2 and Bax
A number of different protocols were attempted to produce specific p-p38 MAPK, Bcl-2 and Bax bands as shown in Figure 4.3. Once transfer was completed, PVDF membrane was placed in blocking solution of either 3%
w/v BSA or 5% w/v non-fat Milk in tris buffer saline-Tween (TBS-T) (150 mM sodium chloride, 20 mM Tris base pH7.4) containing detergent (Tween-20) at concentrations of 0.1% v/v overnight at 40C. The membrane was then incubated with p38 MAP kinase rabbit Ab (diluted in TBS-T,1:2000) or phospho-p38 MAP kinase rabbit mAb (diluted in TBS-T ,1:5000), anti-human Bcl-2 rabbit Ab (diluted in TBS-T,1:2000) or anti-Bax rabbit antibody (diluted
144
in TBS-T,1:2000) overnight with gentle agitation at 40C. The following day, the membranes were washed on a shaker 3 times for 10 min with 10ml TBS-T and then incubated with horseradish peroxidise HRP- conjugated donkey anti rabbit IgG (1:5000) for 1hr at room temperature with shaking. Membrane was washed as previously described then prepared for ECL detection.
145
Figure 4.3: Flowchart representing developmental work that led to the final data of Western blot presented.
4.2.2.1.6.6. Detection of protein by enhanced chemiluminescence (ECL) Following the final wash, the membrane was incubated in equal amounts of ECL solutions, ECL1 (250 mM luminol, 90 mM p-coumaric and 1 M tris base pH 8.5) and ECL 2 (30% H2O2 and 1 M tris base pH 8.5) for 3 min with gentle agitation and then placed in developing cassette, covered with saran wrap
146
excluding air bubbles. Kodak X-ray film was exposed to the membrane in the dark for 5 to 10 min. Film was immersed in developing solution for approximately 3 min until bands were visible, then it was washed with tap water before immersing it in fixing solution until the film became translucent.
Film was rinsed thoroughly with tap water and left to air dry.
All Western blotting experiments were performed on at least 3 independent samples and densitometric analysis of ECL exposure was performed using ImageJ 1.48 software from NIH.
4.2.2.1.6.7. NAC treatment
The antioxidant NAC was used to investigate the role of ROS generation in p38 MAP kinase activation. HUVEC were pre-treated with 2 mM NAC and then cultured for 60 min and 2hr in complete endothelial cell growth medium containing 50 µg/ml IV iron sucrose or IV FCM. HUVEC from the same donors cultured for 3hr in complete endothelial growth medium alone or with 2 mM NAC and were considered as control.
4.2.2.1.6.8. P38 MAPK inhibition
SB203580, a specific inhibitor for p38 MAPK was used to inhibit the phosphorylation of p38 MAP kinase (Gum et al., 1998). HUVECs were cultured to reach 80-90% confluence. Cells were pre-treated with p38 MAPK inhibitor, SB203580 at a final concentration of 10 µM for 1hr. Then cells were treated with IV iron sucrose for 24hr. HUVEC treated with SB203580 alone were considered as a control.
147 4.3. RESULTS