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Meiosis 2 Profase 1Metafase 1 Anafase
Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) was carried out using pre-cast, 15-well NuPAGE® Novex® 4-12% Bis-Tris polyacrylamide gels in the XCell SureLock™ Mini-Cell electrophoresis system, with 800 mL 1x MOPS running buffer (0.05 M MOPS, 0.05 M Tris, 1 mM EDTA, 0.1% (v/v) SDS). The Mini-Cell running unit was loaded with 1 or 2 pre-cast gels, placed into the tank and secured into position by locking the tension wedge. The loading chamber was then filled with MOPS buffer to ensure there were no leaks, after which the combs were removed and each well rinsed out with MOPS buffer to remove any unpolymerised acrylamide. Protein lysates in LDS were prepared as previously described in Section 2.2.4.5, of which up to 25 µL was loaded into each well, with the first well of every gel being loaded with 3 µL of full-range rainbow marker mixed with 22 µL 1x LDS sample buffer. After all the samples were loaded, the remaining MOPS buffer was poured into the tank, the lid was placed on to the running unit and the tank connected to a power supply prior to running the gels at constant amperage of 50 mA, 250 V, 15 W for up to 2 h.
Following electrophoresis, the Mini-Cell was disassembled, the plastic cassettes containing electrophoresed NuPAGE® gels were opened and the stacking gel and ‘foot’ of the gel discarded. The resolving gel was then allowed to equilibrate in 25 mL 1x Tris-Glycine transfer buffer (12 mM Tris, 96 mM glycine, 20% (v/v) methanol) prior to transferring the proteins onto nitrocellulose membrane.
2.2.9.2 Phos-tag™ SDS-PAGE
The Phos-tag™ Acrylamide reagent was utilised in order to enable specific monitoring of phosphorylated proteins. This resulted in a retarded mobility and a distinct upward band shift of the phosphorylated form, from the non-phosphorylated protein, thus permitting the use of a total antibody to detect both the phosphorylated and non-phosphorylated forms.
Phos-tag™ gels were set up and run using the Bio-Rad Mini-PROTEAN® Tetra Cell electrophoresis system, whereby a 1.5 mm glass spacer plate was assembled with a glass short plate in the gel casting unit. Each Phos-tag™ resolving gel was made up in the order as mentioned in Table 2.12, leaving the addition of the APS and TEMED solutions, until just prior to the resolving gel solution being poured between the glass plates.
Chapter Two: Materials and Methods
Table 2.12 Composition of 7% Phos-tag™ SDS-PAGE gels
Resolving gel Stacking gel
Resolving gel buffer
(1.5 M Tris-HCl, pH 8.8, 0.4% (v/v) SDS 2.5 mL N/A
Stacking gel buffer
(0.5 M Tris-HCl, pH 6.8, 0.4% (v/v) SDS N/A 2.5 mL
Ultrapure Protogel® solution (30% (w/v) Acrylamide, 0.8% (w/v) Bis-
Acrylamide (37.5:1)) 2.3 mL 1.3 mL
MnCl2 (10mM) 210 µL N/A
Phos-tag™ Acrylamide reagent
(5mM; 3% (v/v) MeOH) 140 µL N/A
dH2O 4.85 mL 6.2 mL
APS
(10% (w/v) in dH2O) 100 µL 100 µL
TEMED 10 µL 10 µL
On top of each gel, 300 µL dH2O was gently pipetted to eliminate air bubbles and ensure an even edge to the top of the gel, and the gel was left to polymerise for 1 h. Following polymerisation, the dH2O was poured off and the excess blotted, before adding the stacking gel (Table 2.12) and immediately inserting a 15-well, 1.5 mm comb. The stacking gel was then allowed to polymerise for 1 h. The Tetra Cell running units were loaded with 2 gels or 4 gels, positioned within the tank and each loading chamber filled with 1x SDS running buffer (0.025 M Tris-HCl, 0.192 M glycine, 0.1% (v/v) SDS; pH 8.3) to ensure there were no leaks. Following this, the combs were removed and each well flushed out with SDS buffer to remove any unpolymerised acrylamide, prior to loading the samples in each well, with the first well of every gel being loaded with 12.5 µL prestained protein marker. After loading all the samples, the remaining SDS buffer was poured into the tank, the lid placed on to the running unit and connected to a power supply, prior to running the gels at constant amperage of 8 mA or 16 mA, 250V, 15 W for 22 h.
The manganese ions present in the Phos-tag™ gels act as a cofactor and enable the formation of a stable complex with the phosphate group of the proteins during electrophoresis. However, after electrophoresis these ions must be eliminated from the gel in order to increase the efficiency at which the proteins can be transferred onto nitrocellulose membrane.
Chapter Two: Materials and Methods
The Tetra Cell was disassembled, the glass plates containing electrophoresed Phos-tag™ gels were opened and the stacking gel and ‘foot’ of the gel discarded. The resolving gel was carefully placed into 25 mL 1x transfer buffer containing 100 mM EDTA and gently agitated for 10 min. Following this, the EDTA transfer buffer was poured off and the gel was gently agitated for a further 10 min in 25 mL fresh 1x transfer buffer.
2.2.9.3 Western blot analysis
Proteins resolved by SDS-PAGE or Phos-tag™ SDS-PAGE, were transferred onto nitrocellulose membrane using the XCell II™ Blot Module. Sponges provided with the blotting unit were pre-soaked in 1x transfer buffer, along with nitrocellulose membrane cut to 7.5 cm x 8.5 cm. The two electrophoresed gels were sandwiched between pre-soaked filter papers cut to 7.5 cm x 8.5 cm, and these two stacks were placed on top of one another but separated by a pre-soaked sponge. This stack in turn was placed upon another pre-soaked sponge within the blotting unit. A further three pre-soaked sponges were placed on top of the stack, followed by the unit lid, after which the blotting unit was placed into the Mini-Cell tank and secured into position with the tension wedge. The blotting chamber was then filled with 1x transfer buffer, the rest of the tank with cold dH2O and the lid was placed on top prior to transferring the proteins at a constant amperage of 140 mA or 125 mA, 300 V, 15 W for 2 h.
Nitrocellulose membranes with transferred proteins were briefly washed with 1x TBS-T to remove any traces of methanol, which would impede antibody binding. Membranes were then incubated, with gentle rocking, in 25 mL of blocking buffer (5% (w/v) BSA in TBST) at room temperature for 1 h to block non-specific antibody binding sites. Membranes were then incubated with the appropriate primary antibody diluted in 2% (w/v) BSA in TBS-T (as described in Table 2.1 and Table 2.2) at 4ºC for 16 h with gentle agitation. Following incubation with the primary antibody, membranes were washed six times with 1x TBS-T for 10 min with gentle rocking, prior to incubation with the appropriate horseradish peroxidase (HRP)-linked secondary antibody diluted in 2% (w/v) BSA in TBS-T (Table 2.3) at 4ºC for 1.5 h, with gentle agitation. Membranes were then washed for 10 min each time – five washes with 1x TBS-T, followed by a wash with 1x TBS, prior to incubating the membrane with a 1:1 mixture of enhanced chemiluminescence (ECL) reagent 1 and reagent 2 at room temperature for 2 min. The membranes were then encased in a plastic sleeve within an X-ray film cassette and taken into a darkroom before exposing them to X- ray films for various lengths of time, developing and fixing the films.
Chapter Two: Materials and Methods
2.2.9.4 Quantification of protein expression by densitometry
Developed X-ray films were scanned using an Epson® 4490 photo scanner and the image imported into ImageJ (http://imagej.nih.gov/ij/). The image was grey-scaled and the colour inverted to remove any background interference, after which a box set to the size of the largest band was positioned over each band to measure the relative intensity (a setting within the program). To correct for protein loading, the quantified protein of interest band was then related back to its corresponding, quantified Actin band. The untreated control sample in each experiment was assigned an arbitrary value of 1.0, to which bands from all other conditions were relatively expressed.
2.2.9.5 Statistical analysis
Densitometric values from Western blot experiments that had been repeated three or more times were subjected to statistical analysis. A repeated measures ANOVA was performed in SPSS 20 on densitometric values to determine the overall significant difference in the means of the experimental conditions. In the case of sphericity violation (p<0.05 in Mauchly’s Test of Sphericity), a Greenhouse-Geisser correction was utilised. A post hoc test using the Bonferroni correction allowed for pair-wise comparison of each experimental condition to the basal control condition, in order to determine which specific means significantly differed. The means are presented as bar charts with standard deviation bars. The significance levels p<0.05, p<0.01 or p<0.001 were applied accordingly to each experiment and any significant differences were indicated with asterisks.
Chapter Three: Characterisation of ERK5 activation