Materials and Methods
2.10 Protein extraction for gel electrophoresis
2.9.1 Protein extraction from A. mellifera adults and pupae
Three adult/pupal stage A. mellifera, depending on experiment, were crushed in a sterile pestle and mortar to a fine powder using liquid nitrogen.
Care was taken before hand to ensure the samples chosen were of the same age and the same level of parasitization. Samples were resuspended in 3mls of ice cold lysis buffer (20mM PIPES, 5mM NaCl, 0.2% Triton X 100, pH 7.2) and a protease inhibitor cocktail (Pepstatin A (10μg/ml), Aprotinin (10μg/ml), Leupeptin (10μg/ml), Tosyl-lysylchloromethane (10μg/ml), and left mixing slowly at 4oC for two hours to achieve maximum lysis of all remaining cellular structures and breakdown of the large storage proteins that are present in the pupal A. mellifera. The samples were spun at 9000 g for 5 minutes to pellet cellular debris, and the supernatants removed to fresh tubes. For the pupal A. mellifera samples, the samples were allowed to rise to room temperature before centrifugation. The spin step at 9000 g for 5 minutes was carried out at room temperature also. This allows the large amount of lipids present in the young A. mellifera to rise to the top of the supernatant, allowing it to be removed using a fine, sterile spatula. Protein samples were then diluted 1 in 10 in sterile PBS, and kept on ice until quantification by Bradford assay (2.9.4). Proteins samples were quantified to a concentration of 1μg/1μl for 1 dimensional electrophoresis and placed in solubilisation buffer or acetone precipitated according to section 2.9.5 to a concentration of 400μg for 2-Dimensional electrophoresis.
2.9.2 Protein extraction from A. mellifera pupal stage haemolymph
Haemolymph was extracted from six purple eye stage A. mellifera pupae per sample set. Haemolymph was removed using a microcapillary tube inserted laterally along the abdominal section of the bee. Approximately 2-3μl of haemolymph was obtained from each pupa and pooled together in 50μl of ice cold PBS per sample set. Once all six pupae were bled per sample set, the samples were centrifuged at 4oC and 8000g for 5 minutes, brakes off, to pellet haemocytes and other cellular debris. Supernatant was removed to a fresh tube. A further 100μl of ice cold PBS was added per sample, and set aside on ice for quantification by Bradford assay.
2.9.3 Protein extraction from V. destructor
V. destructor adult female mites (0.04 grams) were used per sample set, from colonies with a known level of resistance to Bayvarol. Samples were collected following Autumn treatment from the sticky inserts on the floor of the hive (Figure 2.8). Varroa were isolated from the inserts using a fine tip tweezers and frozen until further use. Varroa were crushed in a miniature pestle and mortar using liquid nitrogen to a fine powder, before resuspension in lysis buffer containing a cocktail of protease inhibitors (see section 2.10.1).
Samples were left mixing at 4o C for two hours, before centrifugation at 9000 g for 5 minutes to pellet cellular debris. Supernatant was then removed to a fresh tube, and kept on ice until quantification by Bradford assay. Protein samples were quantified to a concentration of 1μg/1μl for 1-Dimensional electrophoresis and placed in solubilisation buffer or acetone precipitated according to section 2.10.5 to a concentration of 400μg for 2-Dimensional electrophoresis.
Figure 2.8: Example of a sticky insert from the floor of a hive for the collection of Varroa mites.
The mites are indicated by the arrows, and were collected using a fine tip tweezers.
2.9.4 Bradford assay for protein quantification
Sterile PBS was used to make a serial dilution of bovine serum
albumin and this was used to make a range of standards (0.05-1.5mg/mL). All samples used were diluted in sterile PBS. Twenty µL of sample were placed in a 1mL cuvette. To this 980µL of Biorad Bradford protein assay reagent (Bio-Rad, Hercules, California, USA) was added. Samples were inverted to mix and then allowed to incubate for 5 minutes at room temperature before being read in an Eppendorf Bio-photometer. The quantity of protein was based on the OD590nm readings of the standards as in Figure 2.9.
2mm
Figure 2.9: Standard curve of Bovine serum albumin used to determine unknown protein concentrations
2.9.5 Acetone precipitation of protein samples
Acetone precipitation was used to concentrate protein from a dilute sample volume and also to help purify. The required volume of protein which corresponded to a calculated quantity of protein from Bradford assay quantification (section 2.10.4) was aliquoted to a pre-chilled eppendorf tube and 100% ice-cold acetone was added to the tube at a ratio of 1:3 (sample volume: 100% Acetone). Protein was left to precipitate overnight at -20o C and centrifuged at 13000g for 10 minutes to pellet protein. All pellets were placed upside down to air dry for 5 minutes following removal of acetone.
2.10 1-Dimensional and 2-Dimensional SDS PAGE gel electrophoresis
2.10.1 0.5M Tris-HCl
Tris–HCl (1.5 M) was prepared by dissolving 36.3 g Trizma Base, (Tris Base) in 170ml deionised water and adjusted to pH 8.9 using HCl. Following pH adjustment, 1.5 M Tris – HCl was filter sterilised through a 0.22 μm cellulose filter (Millipore) and stored at 4OC.
2.10.2 1.5M Tris-HCl microcentrifuge tube and filled up to 1ml with deionised water and vortexed briefly to achieve solubility. APS (10% w/v) stocks were frozen.
2.10.4 10x running buffer for electrophoresis
Running buffer (10X), (electrode buffer), was prepared by adding Tris Base 30 g/l, Glycine 144 g/l and SDS 10 g/l to distilled water filled up to 1000ml mark and the mixture was stirred until the solution was solubilised.
Electrode running buffer (10X) stock was diluted to 1X concentration by making 1/10 dilution with distilled water when required.
2.10.5 10% Sodium Dodecyl Sulphate
Sodium Dodecyl Sulphate (SDS), (10% w/v) was prepared by placing 5 g SDS into a 50ml falcon and filling up to 50 ml mark with deionised water and vortexed until all SDS had been solubilised. The solution was stored at room temperature.
2.10.6 5X Solubilisation Buffer for 1-Dimensional gel electrophoresis
Solubilisation buffer was prepared by dissolving the following constituents:
Glycerol 8ml
2.10.7 Preparation of SDS-PAGE mini gels
All glass plates were washed in warm soapy water and cleaned thoroughly with 70% (v/v) ethanol prior to use. SDS–PAGE minigels (cast using the Mini- Protean II gel casting apparatus), were made of acrylamide with 12.5% Bis-acrylamide in all experiments.
These volumes were sufficient to make 3 minigels and volumes were adjusted accordingly where a larger volume was required.
Stacking gel composition
Combs were placed in the gel matrix before it set to create wells for sample loading. These volumes were sufficient to make 2 minigels and volumes were adjusted accordingly where a larger volume was required.
2.10.8 Electrophoresis of 1-Dimensional minigels
SDS-PAGE gels (12.5% Acrylamide) were immersed in 1X running buffer. Samples at a concentration of 20μg/20μl were loaded to each lane using a sterile tip. The gels were electrophoresed at 40V initially and the voltage was increased to 60 V once the protein had travelled sufficiently through the stacking gel. Once the blue tracking dye had moved to the bottom of the gel, the gels were transferred to a clean staining dish.
2.11 2-Dimensional Isoelectric focussing and SDS-PAGE electrophoresis preparation and execution
2.11.1 Isoelectric Focussing Buffer (IEF)
IEF was prepared by dissolving the following constituents:
Urea 8M
Triton X-100 1% (v/v)
CHAPS 4% (w/v)
Tris HCL 10mM
Thiourea 2nM
DTT, 0.2μg per ml of IEF and 2μl of pH 4-7 ampholytes were added per sample prior to use
2.11.2 Equilibration Buffer
Equilibration buffer was prepared by mixing the following constituents:
Tris Base 50nM
Urea 6M
SDS 2% (w/v)
Glycerol 30% (v/v)
The solution was adjusted to pH 6.8 and aliquoted in 40 ml volumes prior to storage at -200 C. For equilibration the buffer was modified as either reducing or alkylating. For reduction, DTT (0.01 g/ml) was added and dissolved thoroughlFor alkylation, IAA (0.025 g/ml) was dissolved thoroughly in the buffer.
2.11.3 Agarose sealing solution
Agarose 1% (w/v)
1X running Buffer 100ml
Bromophenol blue 0.5% (w/v)
Components were mixed together and microwaved until molten, allowing to cool to hand warm before using on gels.
2.11.4 Gel preparation for 2-Dimensional electrophoresis
Glass plates were washed thoroughly with warm soapy water, rinsed and dried with 70% (v/v) ethanol to remove any residual contamination left on the glass. Front glass plates were 200 mm wide and were 200 mm in length, and 223 mm in length at the back. Gels (12.5 % acrylamide) were poured to approximately 190 mm x 160 mm in surface area and 1.5mm thick.
Tris – HCl (1.5 M) 60ml
Deionised water 76ml
Bis-Acrylamide (30% w/v) 100ml
SDS (10% w/v) 2.4ml
APS (10% w/v) 1.5ml
TEMED 0.5ml
2.11.5 2-Dimensional gel electrophoresis
Protein samples (extracted as described in section 2.9.2 and 2.9.3) were
10mins and air dried in preparation for isoelectric focussing. Pellets were resuspended in 100μl of IEF buffer (containing 0.2μg DTT per ml of IEF, and 2μl of pH 4-7 ampholytes per sample) and allowed to resuspend fully by using both vigorous vortexing and sonication in the water bath for 5 minutes.
A further 50μl of IEF, coloured blue using a few grains of bromophenol blue, was added per sample. The sample was briefly vortexed and applied to a 13cm Immobline DryStrip pH 4-7 (G.E. Healthcare). Prior to isoelectric 3. 8000volts Gradient 5 hours 4. 8000volts Step and Hold 8 hours
The second dimension of protein separation was achieved by placing the gels in a Protean PlusTM DodecaTM Cell gel rig with horizontal electrophoresis cells. The chamber was filled to mid-way on the top grey glass plate spacer of all the gel glass plates with 1X electrode running buffer to ensure the buffer covered the acrylamide gel level for proper running. Gels were initially electrophoresed for 1 hour at 1 Watt per gel, and then increased to 1.5 Watts per gel if left overnight provided the gel rig coolant system was kept below 10oC to prevent faster protein migration. The following day gels were increased from 1.5-2.5Watts per gel depending on how far and fast the blue tracking dye line migrated. Gels were monitored at regular intervals to assess the level of electrophoresis and once complete were transferred to a staining dish which was previously washed with soap and 70% ethanol to prevent any contamination to the gels during staining.
Figure 2.10 Isoelectric focussing machine used for all first dimension separation during 2-Dimensional SDS-PAGE gel electrophoresis