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To make a mixed-membrane preparation, cells were collected from the culture dishes (60mm-Sf9, 90mm-HEK 293) by gentle tapping and pipetting. Cells were then counted using a hemocytometer and then re-suspended in hypo-osmotic buffer (see Appendix A section 3) at a density of 1x106 cells (HEK 293) or 2x106 cells (Sf9) per ml of buffer. Cell aliquots of 10-15ml were homogenised using a glass homogeniser kept on ice (around 10 even strokes), and each aliquot was then passed at least 10 times through a 23G gauge needle attached to a 10ml syringe. The homogenate was centrifuged at 2700rpm (1500g) and 4oC for 15 minutes to remove broken cells and nuclei. The supernatant was carefully removed and then centrifuged at 28,000rpm (100,000g) in a Beckman Optima–L90K centrifuge, using a 50.2 Ti rotor, for 1h at 4oC. The pellet was then transferred to a 1ml glass homogeniser kept on ice, resuspended in 0.4M sucrose, 20mM HEPES solution (20µl per 1x106 (HEK 293) cells or 2x106 (Sf9) cells) and homogenised to give a fine suspension. Mixed- membrane preparations were first flash frozen in liquid nitrogen and stored in 100µl aliquots in a -80oC freezer.
4.2.3.2 Protein assays
A micro BCATM (bicinchoninic acid assay) protein assay (ThermoScientific, MA, USA) was used to quantify protein. The mixed-membrane sample was diluted 1:50, 1:100, 1:200 in double distilled water, mixed well, and 100µl of the diluted protein was loaded into a 96 well (flat bottom) plate, either in duplicate or triplicate. 200µl of working reagent (a mixture of kit reagents A, B and C) was then added to each well and the plate incubated in the dark for 15 minutes. The absorbance of each well at 595nm was measured using a UV max kinetic micro-plate reader (Molecular Devices, CA, USA). The protein concentration of each mixed-membrane preparation was then calculated by fitting the average absorbance values of each duplicate or triplicate sample onto the BSA (bovine serum albumin) standard curve.
Alternatively, a Bradford reagent (Bio Rad, CA, USA) assay was used. The mixed- membrane preparation was diluted 1:2, 1:4, 1:8, 1:16 in double distilled water and measured against a set of BSA standards in a 96 well (flat bottom) plate. 200µl of Bradford reagent and 5µl of the diluted mixed-membrane sample were added to each well. All concentrations were prepared in duplicate. Readings were taken after a 10 minute incubation by measuring the absorbance at 590nm, using a SpectraFluor Plus
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(Tecan, Dorset, UK) micro-plate reader, and the protein concentration in each well was determined by the Magellan™ data analysis software (Tecan) associated with the plate reader.
4.2.3.3 Radio-ligand binding assays
Two different methods were used for the radio-ligand binding assay experiments; a glass micro-fibre GF/B filter disc (GE Healthcare-Whatman®, Little Chalfont, UK) assay, which was used for testing the expression levels of the RyRs, and a 96 well GF/B filter plate (PerkinElmer, MA, USA) based assay which was used to determine the binding kinetics of ryanodine to the receptors.
The glass micro-fibre filter disc assay protocol was as follows: The binding buffer (recipe in Appendix A, section 5.1) was made up on the day of the experiment. Experiments were done in duplicate or triplicate for hot (total binding) and cold (non-specific binding) reactions. For the hot reaction, samples were set up in 3ml vials (VWR, PA, USA) in a total volume of 500µl, consisting of 100µg of the mixed-membrane preparation, 20µl of [3H] ryanodine (0.1mCi/ml) (PerkinElmer, MA, USA) (final concentration of [3H] ryanodine 8nM) and binding buffer up to 500µl. The cold reaction had 100µg of mixed-membranes, 20µl of [3H] ryanodine, 5µl of cold ryanodine (final concentration 5µM) and binding buffer up to 500µl. Samples were thoroughly mixed by vortexing and incubated in a 37oC water bath for 90 minutes. The filter discs were pre-soaked in binding buffer and, after the sample incubation was completed, the samples were loaded onto the glass micro-fibre filters using a vacuum manifold. The glass vials were then washed with 500µl of binding buffer to recover any residual sample. Following sample binding, filter discs were washed with an additional 500µl of binding buffer. After the vacuum pump filtration was completed all discs were transferred to individual scintillation vials containing 5ml of Ultima gold™ MV scintillation fluid (PerkinElmer, MA, USA), and loaded onto a Packard Bioscience (now PerkinElmer) 2100TR TriCarb Liquid Scintillation Analyzer for counting. Obtained dpm values were then processed to calculate the amount of specific binding using Office Excel 2010 (Microsoft, CA, USA).
The 96 well filter plate assays were carried out as follows: the mixed-membrane preparation was diluted in buffer A (all the buffers recipes can be found in Appendix A, section 5.2) to a final concentration of 0.25mg/ml. Both total and non-specific
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reactions were set up in a 96 well flat bottom microtiter plate in a total volume of 250µl. For each reaction 48µl of Buffer B containing 0.01% Pluronic (Sigma, MA, USA) was added together with 2.5µl of DMSO (for total reaction) or 1mM ryanodine (final concentration 10µM) in DMSO (for non-specific reaction), 100µl of diluted mixed-membrane preparation and 100µl of diluted [3H] ryanodine (to the desired final concentration). Empty wells on the plate were filled with buffer B and diluted (old) membrane preparation. The plates were shaken gently and left to incubate at room temperature for 2h. The 96 samples were then loaded onto the filter plate (with each well pre-soaked with 50µl of 0.1% polyethylenimin in wash buffer) using a 96 well plate harvester (Brandel, MD, USA). The filter plate was then washed 3 times with 250µl wash buffer and left overnight at room temperature to dry. Each well on the plate was then filled with 50µl MicroScint™-O (PerkinElmer, MA, USA) and the filter plate loaded into a TopCount NXT™ Micro-plate Scintillation counter (PerkinElmer, MA, USA). Specific binding and binding kinetics values (equilibrium dissociation constant Kd and Binding capacity/receptor
density Bmax) were calculated using GraphPad Prism v5.5 (GraphPad, CA, USA).
4.2.3.4 Western Blotting
Mixed-membrane preparations from HEK 293 cells transfected with P. xylostella or M. persicae RyR-eGFP fusion constructs were run on 5% (w/v) polyacrylamide gels (recipe in Appendix A, section 4) and blotted onto nitrocellulose membranes using an iBlot 7 minute blotting system (Life technologies, CA, USA) following the manufacturer’s recommended protocol. The nitrocellulose membranes were then blocked with 5% (w/v) powdered skimmed milk (VWR, PA, USA) in TBS-T (recipe in Appendix A, section 4) for 1h at 4oC, washed with TBS-T and then incubated overnight at 4oC with primary mouse monoclonal anti-eGFP antibody (Santa Cruz biotechnology, TX, USA) at a 1:5000 dilution in 1% (w/v) powdered milk/TBS-T. After removal of unbound primary antibody with 3 washes of TBS-T buffer, the blot was incubated for two hours with 1:5000 diluted secondary goat anti-mouse horseradish peroxidase conjugated antibody (Sigma, MA, USA) in milk/TBS-T After a further 3 washes with TBS-T buffer, the antibody attached peroxidase activity was detected by breakdown of a luminol (ECL) substrate (GE Healthcare, Little Chalfont, UK) and exposure of the blot to X-ray film. The protein was
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visualised after a 30 minutes exposure by developing the X-ray film using an X4 Automatic X-ray Film Processor (Xograph, Stonehouse, UK).