To determine the expression levels of cellular proteins as a result of different experimental conditions (see individual results sections), HPC or cells from AML- derived cell lines (5 million cells for HPC and Mv4;11 and 2 million cells for THP-1
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and NOMO-1 derived cells) were centrifuged at 270g for 5 min and washed in tris- buffered saline (TBS) (composed of 1M Tris-HCl, 130mM NaCl (both Sigma)) (10mL), followed by centrifugation at 270g for 5 min and the supernatant removed and flash frozen in liquid nitrogen. Frozen cells were stored at -80oC or used below. 2.9.1 Whole cell lysate extraction
Frozen cell pellets (2.9) were thawed on ice in the presence of DNase (1µg) (Sigma). Cells were treated with lysis buffer (composed of 10 mM HEPES-KOH (Invitrogen), 10 mM 2-mercaptoethanol (Sigma), 1mM magnesium acetate (Fisons Scientific Equipment), 0.5 mM EDTA (Sigma), 0.5 mM EGTA (Sigma), 0.25 M sucrose (Sigma), 1 mM Na3VO4 (Sigma),1 complete mini EDTA protease inhibitor tablet per 10 mL (Roche), 1% v/v x100-Triton (Sigma)) (2 x 107 cells/mL), vortexed, incubated on ice for 30 min and subjected to occasional vortexing. The cell lysate was
centrifuged at 12,782g for 5 min at 4oC and the protein concentration of the supernatant determined by Bradford assay (2.9.3).
2.9.2 Nuclear/Cytosol Extraction and Fractionation
THP-1 cells centrifuged at 270g for 5 min, washed in TBS (10mL) as above and the supernatant removed leaving washed cell pellets for nuclear/cytosol fractionation using a kit from Biovision (California, USA), according to the manufacturer’s instructions. Briefly, cell pellets were resuspended in proprietary cytosol extraction buffer A (100µL/million cells) containing protease inhibitor cocktail (2µL) and dithiothreitol (1µL). Cells were vortexed (15s), incubated on ice for 10 min, cytosol extraction buffer B (5.5µL/million cells) added, vortexed (5 sec) and incubated on ice for a further minute. Cells were then vortexed for 5s, centrifuged at 12,782g for 8 min at 4oC and the supernatant (containing the cytosolic extract) collected and stored at -800C. The remaining cell pellet was washed in PBS (Invitrogen) containing MgCl2 (0.5mM) (Sigma), centrifuged at 12,782g for 3 min at 4oC, the supernatant discarded and the pellet snap frozen in liquid nitrogen. The samples were subjected to three rapid freeze/thaw cycles in liquid nitrogen followed by addition of
Benzonase (25U/µL) (Novagen) at 2µL/million cells. The samples were kept on ice for 60 min before TEAB lysis buffer was added at 10µL/million cells. The samples were kept on ice for a further 30 min, subjected to occasional vortexing, before being centrifuged at 12,782 x g for 10 min at 4oC and the supernatant stored at -80oC.
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Protein quantification of the nuclear and cytosol extracts was performed, as described below (2.9.3).
2.9.3 Protein concentration quantification
Bradford’s reagent (Sigma) was diluted 1:1 with tissue culture grade sterilised water (Hameln) and 190μl added to duplicates of the cell lysate sample (10μl), diluted 20- fold with tissue culture grade sterilised water (Hameln), in a 96-well flat-bottomed plate. The plate was equilibrated to RT for 10 min, then absorbance measured (590nm) using a spectrophotometer (ASYS Hitech, GmbH, Eugendorf, Austria) and compared with BSA protein standards (0-10 μg/mL) assayed in duplicate on the same plate. Protein concentration of the cell lysate was determined from the linear part of the standard curve as described above (2.7). Variation between duplicates of the measured absorbance of the protein standards was limited to a co-efficient of variation (CV) of 5% and variation between cell lysates duplicates limited to 10% CV.
2.9.4 Protein electrophoresis and electroblotting
Proteins were separated by SDS-PAGE using Nu-PAGE 4-12% Bis-Tris Mini Gels (Invitrogen) and electroblotted according to the manufacturer’s instructions using proprietary Nu-Page reagents purchased from Invitrogen.
Prior to separation by electrophoresis, denaturation and reduction of disulphide bonds was performed. Protein extracts, from the cell lysates, were incubated at 70oC for 10 min in the presence of Nu-Page x1 LDS buffer (containing lithium dodecyl sulphate) (Invitrogen) and 50mM DTT (Invitrogen). To ensure an equal volume of protein across samples in the final denatured protein extracts, the volume of protein added was adjusted with tissue culture grade sterilised water (Hameln), according to the value determined from the Bradford’s assay (2.9.3), to a final concentration of 1μg/μL.
Bis-Tris gels were placed in a X-Cell IITM mini-cell containing x1 running buffer (composed of 50mM 3-(N-morpholino)propanesulfonic acid, 50mM tris base, 0.1%
w/v sodium dodecyl sulphate and 0.25% antioxidant, and proteins loaded (20μg) on
to the gels alongside x1 LDS buffer, containing 5% v/v MagicMark XPTM, to allow visualisation of protein molecular weight. Following 200 volts electrophoresis for 50 min gels (x1 or x2) were placed in a X-Cell IITM blotting module, next to a
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nitrocellulose membrane in the presence of x1 transfer buffer containing methanol (10% v/v for 1 gel or 20% v/v for 2 gels) and electroblotted at 30 volts for 60 min. Electroblotted membranes were washed in dH2O on a plate shaker for 5 min (x2) and proteins visualised using Ponceau S solution (1.3mM Ponceau S, 0.83M ethanoic acid) (Sigma), in order to facilitate cutting of membrane if required. Membranes were subsequently washed as above, incubated on a plate shaker for 60 min in TBS- T (TBS buffer with 0.1% v/v Tween-20) and 2% w/v milk powder (Marvel), then washed for 15 min in TBS-T and then a further three times for 5 min in TBS-T. Membranes were subsequently used for immunoblotting.
2.9.5 Immunoblotting
Membranes were incubated overnight at 4oC, in 10 mL TBS-T containing 2% w/v milk powder, containing antibodies at concentrations described in (Table 2). Membranes were washed with 20mL of TBS-T for 15 min on a plate shaker
followed by three subsequent 20 mL washes for 5 min. Subsequent incubation of the membrane occurred at RT on a plate shaker in the presence of TBS-T containing 1%
w/v milk powder and secondary anti-mouse or anti-rabbit horse radish peroxide
conjugated antibody (GE Healthcare), diluted 1:5000, for 60 min. Following incubation, membranes were washed four times in TBS-T as above and target proteins were visualised using the ECL Advance Detection Kit (GE Healthcare), according to the manufacturer’s instructions. Chemiluminescence was detected using a LAS-3000 image analyser (Fujifilm UK Ltd) and analysed using AIDA Image Analyser Version 4.19 (Fujifilm UK Ltd). To confirm equal loading of proteins the nitrocellulose membrane was washed and reprobed as above using anti- β-actin or anti- α-tubulin for whole cell protein extraction, or anti- β-actin for cytosol protein extraction and anti-histone H1 for nuclear protein extraction (Table 2). Relative changes in protein expression were measured using AIDA Image Analyser software (Raytest, Straubenhardt, Germany). Briefly, the area of a region of interest was determined and the pixel density of this region compared with equivalent areas of interest. The pixel density of equal sized areas around the comparative areas of interest was used to determine the background signal, which was subtracted prior to comparison, and the relative pixel density levels normalised to the control sample.
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