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2.6.1 Nuclear/Cytoplasmic Protein Extraction

Protein for western blotting was extracted from transfected cells and fractionated using the NE- PER Nuclear and Cytoplasmic Reagents (Pierce) according to the manufacturer’s instructions, but altering the volumes. Media was removed from cells in a 12 well plate and a cell suspension made in 500 µL fresh, unsupplemented DMEM. Cells were pelleted by centrifugation at 18000 x

g and the media removed and replaced with 50 μL of ice cold CER I buffer, with Protease Inhibitor (cOmplete, EDTA-free Protease Inhibitor, Roche) and Phosphatase Inhibitor (PhosStop Phosphatase Inhibitor Cocktail, Roche) added at final concentrations of 1X. Cells were vortexed thoroughly and incubated for 10 minutes on ice. Following incubation, 2.25 μL of CER II was added and cells were again vortexed followed by incubation on ice for one minute and a second vortex. Cells were then pelleted by centrifugation at 18000 x g for 15 minutes at 4 o_{C, and the}

resulting cytoplasmic supernatant transferred to a new 1.5 mL Eppendorf tube. 25 μL of NER buffer, again containing 1X Protease Inhibitor and Phosphatase Inhibitor, was added to the cell pellet. The cells were resuspended by vortexing thoroughly and the samples incubated on ice for 40 minutes, vortexing every 10 minutes to mix. The samples were pelleted by centrifugation at 18000 x g, for 25 minutes at 4 o_{C and the resulting nuclear supernatant transferred to a new}

1.5 mL tube.

To prepare the cytoplasmic and nuclear fractions for Sodium Dodecyl Sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), DTT (dithiothreitol; Sigma-Aldrich, at 20X) and NuPAGE LDS Sample Buffer (Invitrogen; 4X) were added to the cell lysates to give a final concentration of 50 mM and 1X respectively. The samples were heated at 90 o_{C for 5 minutes to denature the proteins.}

Denatured lysates were then either used immediately or stored at -80 o_C. 2.6.2 SDS-PAGE

Protein from cytoplasmic and nuclear cell lysates was separated by molecular weight (MW) on an acrylamide gel. Polyacrylamide 10% running gel mixes (Table 2.8) were made and cast in the Mini PROTEAN® casting apparatus (Bio-Rad) while still liquid. Running gels were overlaid with hydrated butanol (Sigma-Aldrich) and left at room temperature to polymerise for 1 hour. Following polymerisation, the butanol was removed, the running gels were overlaid with a

Table 2.8: Reagents and volumes for a 10% running polyacrylamide gel. Volumes are for one gel of 1.5 mm thickness.

Reagent Volume

40% Acryl:Bisacryl (37.5:1) (Sigma-Aldrich; Cat. No. A7168) 3.13 mL 1.5 M Tris pH 8.8 (Sigma-Aldrich; Cat No. 252859) _{3.13 mL} 10% SDS (Sigma-Aldrich; Cat. No. L4390) _{125.00 µL}

ddH20 6.00 mL

10% APS (Sigma-Aldrich; Cat. No. A3678) _{125.00 µL} TEMED (Sigma-Aldrich; Cat. No. T9281) _{12.50 µL}

Table 2.9: Reagents and volumes for a 3.75% stacking polyacrylamide gel. Volumes are for one gel of 1.5 mm thickness.

Reagent Volume

40% Acryl:Bisacryl (37.5:1) (Sigma-Aldrich; Cat. No. A7168) 235.00 µL 0.5 M Tris pH 8.8 (Sigma-Aldrich; Cat No. 252859) _{625.00 µL} 10% SDS (Sigma-Aldrich; Cat. No. L4390) _{25.00 µL}

ddH20 1.59 mL

APS (Sigma-Aldrich; Cat. No. A3678) _{50.00 µL} TEMED (Sigma-Aldrich; Cat. No. T9281) _{5.00 µL}

3.75% stacking gel (Table 2.9) and a comb was inserted to create wells for loading. The gels were again left to polymerise for 1 hour.

Once polymerisation was complete, the gels were placed into a BIORAD Mini PROTEAN® Tetra Cell apparatus filled with 1X SDS-PAGE Running Buffer (192 mM Glycine, 24.9 mM Tris base and 3.47 mM SDS). 5-30 μL of each sample were loaded into the separate wells of the gel. Alongside these samples, 8 μL of PageRuler Prestained Protein Ladder (Fermentas) was added to visualise the protein separation during gel running and to determine approximate size of proteins after western blotting. Gels were run at 100 V for 1.5-2.5 hours to separate the proteins, using the separation of the Protein Ladder as a guide for running time.

2.6.3 Wet Transfer

Once the gel was run to an appropriate extent, the protein was transferred onto a membrane using wet transfer. The stacking portion of the gel was discarded and the running gel placed on a piece of polyvinylidene difluoride (PVDF) membrane, which had been activated in 100% methanol (MeOH). The gel and membrane were sandwiched between four pieces of blotting paper (3 mm chromatography paper, Whatman®) and two sponges within a gel holder cassette, with all components submerged in 1X Towbin’s Buffer (190 mM Glycine, 24 mM Tris base and 20% MeOH) to allow the gel to equilibrate and to reduce air bubbles. The gel holder cassette (containing the gel, PVDF, blotting paper and sponges) was transferred to a mini Trans-Blot Module (Bio-Rad) within a gel tank filled with 1X Towbin’s Buffer, and run at 15 V overnight (~16 hours) to transfer the protein to the PVDF membrane. During this time, the tank was surrounded by ice to minimise heating.

2.6.4 Western Blotting

Once transfer was complete the, PDVF membrane (to which the protein is now bound) was removed from the transfer apparatus and blocked in blocking buffer (5% skim milk [Diploma], 0.02% Tween 20 in PBS) for at least 1 hour with agitation. Following blocking, the membrane was exposed to a primary antibody (Table 2.10) diluted in 3 mL of blocking buffer for 2-2.5 hours. The membrane was then washed with blocking buffer six times, with each wash lasting 5-10 minutes. Following washing, the membrane was exposed to the appropriate Horse Radish Peroxidase (HRP) conjugated secondary antibody (Table 2.11) diluted in 3 mL of the blocking buffer for 1-1.5 hours and then washed with 0.02% Tween in 1X PBS six times, with each wash lasting 5-10 minutes.

The membrane was incubated in SuperSignal West Pico Chemiluminescent Substrate (Pierce) for 5 minutes in darkness. The SuperSignal West Pico Chemiluminescent Substrate contains a

Table 2.10: Primary antibodies used in western blotting.

Target Protein Company; Cat. No. Species Dilution used

GAPDH Abcam; ab8245 Mouse, monoclonal 1/1000 HA-probe (Y-11) Santa Cruz; sc-805 Rabbit, polyclonal 1/200 Tata binding protein (TBP) Abcam; ab818 Mouse, monoclonal 1/2000 V5 Invitrogen; R960-25 Mouse, monoclonal 1/3000

Table 2.11: Secondary antibodies used in western blotting.

Species Target Species Company; Cat. No. Dilution used

Rabbit Mouse Invitrogen; 616520 1/5000 Donkey Rabbit Invitrogen; A21206 1/500

substrate upon which the HRP tag of the secondary antibody can act to produce a chemiluminescent signal. Following incubation, the membrane was immediately exposed to a charge-coupled camera via the ImageQuant™ LAS4000 biomolecular imager (GE Healthcare Life Sciences) for 10 seconds - 10 minutes (dependent on the experiment and antibody used) and a digital image produced.