2.5.1 Whole Cell Lysis
Lysis of fibroblast or myoblast pellets was performed on ice. The cell lysate buffer comprised 50mM Tris-HCl, 130mM NaCl, 2mM MgCl2, 1% NP-40 (v/v) and 1X EDTA-protease
inhibitor cocktail tablet. Cell lysate buffer was prepared in 10ml stocks and stored at -20OC in 200µl aliquots. Immediately before use, 1mM PMSF was added to the lysate buffer.
Dependent on the pellet size, 50-100µl cell lysate buffer was added to each pellet and mixed by pipetting. All samples were vortexed thoroughly for 30 seconds, then centrifuged at 4OC,
2,300rpm for 2 minutes. After centrifuging, the supernatant was transferred to a clean 1.5ml eppendorf tube then rapidly frozen in liquid nitrogen for storage at -80oC.
2.5.2 Muscle Homogenisation
Frozen skeletal muscle was homogenised for western blot studies using a protease buffer prepared from RIPA buffer. Liquid nitrogen was added to a pestle and mortar for rapid cooling, then 10-20mg frozen control or patient muscle was added to the mortar. Using the pestle, the muscle was crushed to obtain a powder with further liquid nitrogen added to cool the muscle, pestle and mortar. Once a fine powder was attained, 0.5-1ml protease buffer was added to the tissue. Once frozen, the tissue continued to be crushed until the protease buffer
and tissue were thawed in a homogenate, then pipetted into a clean 1.5ml eppendorf tube. The homogenate was vortexed for 15 seconds then placed on ice for 5 seconds. This was repeated a further four times, then placed on ice for 45 minutes. Next, the homogenate was
homogenised using the either the T25 or T8 Ultra-Turrax homogeniser (IKA) or for 5
seconds, placed on ice for 15 seconds, then repeated. The homogenate was returned to a clean 1.5ml eppendorf tube and centrifuged in a chilled centrifuge at 4OC, 11,500rpm for 10
minutes. After centrifuging, the supernatant was transferred to a clean 1.5ml eppendorf tube and rapidly frozen in liquid nitrogen for storage at -80OC.
2.5.3 Bradford Assay
Protein quantification was performed using a Bradford assay with bovine serum albumin (BSA) to produce a standard curve. BSA stocks were prepared at 1mg ml-1 and stored at - 20OC. The standard curve was prepared in duplicate with BSA calibration points at 0, 2, 5, 10, 15 and 20mg ml-1. Protein samples of unknown concentrations were prepared at 1µl and 2µl dilutions; no duplicates were made. All tubes were vortexed after addition of all the reagents and left at room temperature for 5 minutes. Once the BSA calibration points and protein samples for quantification were prepared, 200µl were aliquoted using reverse pipetting into a 96-well plate. Each protein sample for quantification was aliquoted twice. Plates were then read at 595nm using the SpectraMax M5e Multimode Plate Reader (Molecular Devices).
2.5.4 SDS-Page Sample Preparation
Fibroblast, myoblast and muscle lysates were prepared for western blot loading using 4x or 5x laemmli sample buffer. Hence, the ratio of lysate to sample buffer was 3:1 or 4:1. Prepared samples were heated at 37OC on a dry heat block for 30 minutes. After heating, 0.5µl
benzonase nuclease (Merck Millipore) was added to each sample to degrade residual nucleic acid. Samples were flicked to mix and kept on the laboratory bench at room temperature for a further 15 minutes.
2.5.5 SDS-Page
Polyacrylamide gels were handcast and electrophoresis performed using the Mini-Protean Tetra Cell system (BioRad) or the Mini Electrophoresis Blotting System (Hoefer). Either 10% or 12% polyacrylamide gels were used, depending on the molecular weight of the proteins of interest (Table 2.3). Once the resolving gel was poured, isopropanol was added on top to
produce an even edge until polymerisation. After the isopropanol was washed away, the 3.75% stacking gel was poured on top.
10% Resolving Gel 12% Resolving Gel 3.75% Stacking Gel Acrylamide/Bis-acrylamide, 30%, 29:1 1.667ml 2ml 0.625ml 3.75M Tris-HCl, pH 8.5 0.5ml 0.5ml / 0.5M Tris-HCl, pH 6.8 / / 1.25ml dH2O 2.726ml 2.395ml 3.02ml 10% SDS 50µl 50µl 50µl TEMED 5µl 5µl 5µl 10% APS 50µl 50µl 50µl
Table 2.3 Polyacrylamide Gel Casting Reagents and Volumes. List of reagents and volumes for casting a 10% or 12% resolving gel and the 3.75% stacking gel.
SDS-page electrophoresis was performed in 1X running buffer (25mM Tris, 192mM glycine, 0.1% SDS) with 25-50µg protein per sample as prepared in 2.5.4, plus 10µl Spectra
Multicolor Broad Range Protein Ladder (Thermo Scientific). Electrophoresis was run at a constant 150V using the Bio-Rad Mini-Protean Tetra Cell System or 120V using the Hoefer Mini Electrophoresis Blotting System until the sample buffer dye reached the bottom of the polyacrylamide gel.
2.5.6 Wet Electroblotting Transfer and Blocking
The transfer of proteins from the SDS-page gel to a polyvinylidine fluoride (PVDF)
membrane (Bio-Rad) was performed using wet electroblotting transfer with the Mini Trans- Blot Cell System (Bio-Rad). The SDS-page gel was removed from the gel running system and placed in 1X transfer buffer (25mM Tris, 192mM glycine). The PVDF membrane
(8.5x5.5cm) was activated in 100% methanol for 30 seconds and rinsed in distilled water. A cassette was assembled with the gel and membrane between four cut-pieces (8.5x5.5cm) 3mm thick chromatography paper (Whatman) and two sponges. This was placed in the transfer tank filled with 1X transfer buffer, a magnetic stirring bar and an ice pack. Wet transfer was
performed at 100V for 60 minutes, at 4OC on a magnetic stirrer to keep the transfer buffer cool. Following transfer, membranes were blocked in 5% powdered milk (Marvel) in 1X TBST for 1 hour at room temperature to prevent non-specific binding of antibodies.
2.5.7 Ponceau S Staining
Ponceau S staining solution was used for qualitative visualisation of protein loading on PVDF membranes. Following wet electroblotting transfer, membranes were incubated with Ponceau S solution for 2 minutes at room temperature and rinsed in dH2O until background staining
was removed. After visualisation, membranes were further rinsed in dH2O until all staining
solution was removed.
2.5.8 Primary and Secondary Antibodies
Membranes were probed with primary antibodies diluted in 5% powdered milk in 1X TBST (Table 2.4); either overnight at 4OC or for 2 hours at room temperature. Anti-alpha Tubulin,
anti-β-actin, anti-GAPDH and anti-Vinculin were used as loading controls.
Antibody Dilution Predicted (Observed)
Size, kDa
Host Species
Clonality
Anti-AK2 (ab37594, Abcam) 1:1000 26 Rabbit Polyclonal
Anti-AK3 (ab119058, Abcam) 1:1000 26 Mouse Monoclonal
Anti-alpha Tubulin (ab7291, Abcam) 1:10000 50 Mouse Monoclonal
Anti-ATP5B (ab14730, Abcam) 1:2000 52 Mouse Monoclonal
Anti-beta Actin (A5316, Sigma) 1:10000 42 Mouse Monoclonal
Anti-GAPDH (ab8245, Abcam) 1:10000 40 (36) Mouse Monoclonal
Anti-GMPR1 (ab118751, Abcam) 1:1000 37 Rabbit Polyclonal
Anti-GMPR1 (SAB1101144, Sigma) 1:1000 37 (40-42) Rabbit Polyclonal
Anti-LONP1 (15440-1-AP, Proteintech)
1:1000 100 Rabbit Polyclonal
Anti-MRPL12 (Affinity purified in Lightowler laboratory)
1:500 19 Rabbit
Anti-MTCOI (ab14705, Abcam) 1:1000 57 (40) Mouse Monoclonal
Anti-MTCOII (ab110258, Abcam) 1:1000 26 Mouse Monoclonal
Anti-mtAlaRS/AARS2 (ab197367, Abcam) 1:1000 107 Rabbit Polyclonal Anti-mtGluRS/EARS2 (HPA043633, Sigma) 1:1000 60 Rabbit Polyclonal Anti-mtTyrRS/YARS2 (AP7838a, Abgent) 1:500 60 Rabbit Polyclonal Anti-mtRNA Polymerase/POLRMT (ab32988, Abcam) 1:250 139 (140) Rabbit Polyclonal
Antibody Dilution Predicted (Observed) Size, kDa Host Species Clonality Anti-MT-TFA/TFAM (ab119684, Abcam) 1:1000 29 (25) Mouse Monoclonal
Anti-NDUFB8 (ab110242, Abcam) 1:1000 22 Mouse Monoclonal
Anti-p53R2 (ab8105, Abcam) 1:5000 41 (39) Rabbit Polyclonal
Anti-R1 (sc-22786, Santa Cruz) 1:1000 94 Rabbit Polyclonal
Anti-R2 (sc-10846, Santa Cruz) 1:1000 45 Goat Polyclonal
Anti-RMDN3/PTPIP51 (HPA009975, Sigma)
1:500 62 Rabbit Polyclonal
Anti-SDHA (ab14715, Abcam) 1:1000 70 Mouse Monoclonal
Anti-SLC25A33/PNC1 (ab97820, Abcam) 1:1000 35 Rabbit Polyclonal Anti-SLC25A36/PNC2 (ab154559, Abcam) 1:1000 34 Rabbit Polyclonal Anti-SLC29A1/ENT1 (LS-C96639, Lifespan Biosciences) 1:1000 50 Rabbit Polyclonal Anti-TK1 (H00007083-M02, Abnova) 1:500 26 Mouse Monoclonal
Anti-TK2 (Liya Wang) 1:1000 26 Rabbit Polyclonal
Anti-UQCRC2 (ab14745, Abcam) 1:1000 48 Mouse Monoclonal
Anti-VDAC1/Porin (ab14734, Abcam) 1:10000 39 (37) Mouse Monoclonal Anti-Vinculin/VCL (ab18058, Abcam) 1:2000 130 Mouse Monoclonal
Table 2.4 Primary Antibodies. List of primary antibodies used for western blotting.
Membranes incubated overnight at 4OC were transferred to room temperature for a further 20 minutes of incubation to enhance the signal. Next, membranes were washed five times at 5 minutes in 1X TBST, then probed with horseradish peroxidase (HRP)-conjugated secondary antibodies (Dako) diluted in 5% powdered milk in 1X TBST (Table 2.5) for 1 hour at room temperature.
Antibody Dilution Host Species Clonality
Anti-Goat Secondary (P0449, Dako) 1:3000 Rabbit Polyclonal
Anti-Mouse Secondary (P0260, Dako) 1:2000 Rabbit Polyclonal
Anti-Rabbit Secondary (P0399, Dako) 1:3000 Swine Polyclonal
Table 2.5 Secondary Antibodies. List of HRP-conjugated secondary antibodies used for immunodetection.
After secondary antibody incubation, membranes were washed a further five times for 5 minutes in 1X TBST. Detection of secondary antibodies was performed using either Clarity Western ECL Substrate (Bio-Rad) or SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). A 1:1 ratio of the luminol/enhancer reagent and stable peroxide solution were mixed, added to the membrane surface with the protein side up and incubated in
darkness for 5 minutes. Fluorescence was measured using the ChemiDoc MP Imaging System (Bio-Rad) and analysed using the Image Lab (Bio-Rad) software.