CODIFICACIÓN DE LOS DATOS

Pellets of harvested cells were resuspended in approximately 50µl of lysate buffer per 10mg of wet cell pellet.

Lysate buffer was prepared with 50mM Tris/HCl pH 7.4, 130mM NaCl, 2mM MgCl2,

1mM PMSF, 1% Nonidet P-40, one EDTA-free protease inhibitor tablet (Pierce) and was made up in a 10ml final volume before being aliquoted and stored at -20°C.

Samples were vortexed for 30 seconds and centrifuged at 1000g for 2 minutes to remove nuclei and unbroken cells. The supernatant was then transferred to a fresh microcentrifuge tube, snap frozen in liquid nitrogen and stored at -80°C.

Samples with small cell pellets were resuspended in 15-30µl of lysate buffer and vortexed, but were not subjected to centrifugation in order to prevent loss of material. Instead, 25U Benzonase was added to remove nucleic acids. If any sample for a particular experiment needed to be prepared in this way, then all of the samples were treated in the same manner (samples prepared in this manner will be designated whole cell lysate, whereas samples with nuclei removed by centrifugation will be designated cell lysate). Estimation of protein concentration (2.5.3) was always performed prior to the addition of Benzonase.

2.5.2M

P

Mitochondria were isolated from cells grown in 300cm2 flasks. These were harvested at approximately 80% confluency and the resulting pellet was resuspended in 2ml ice-cold homogenisation buffer (0.6M mannitol, 10mM Tris pH 7.4 and 1mM EGTA) supplemented with 0.1% BSA and 1mM PMSF. The following steps were carried out at 4°C. The cell suspension was transferred to a glass:Teflon Dounce homogeniser and subjected to 15 passes before being centrifuged at 400g for 10 minutes. The supernatant was retained in pre-chilled tubes, and the pellet was resuspended in a further 2ml of supplemented homogenisation buffer and the homogenisation and centrifugation process repeated. The supernatant was again retained and all supernatant tubes were centrifuged at 400g for 5 minutes. The supernatants were transferred to new microcentrifuge tubes and the mitochondria were pelleted at 11,000g for 10 minutes.

The pellet was washed twice in homogenisation buffer lacking BSA and finally resuspended in 70-150µl homogenisation buffer.

To remove cytosolic contamination 1mg of isolated mitochondria were treated with 1µg proteinase K on ice for 30 minutes. Digestion was then inhibited by addition of 1mM PMSF.

2.5.3E

P

C

B

A

Measurement of protein concentration was performed using the Bradford assay. Samples were added to a final volume of 800µl of dH2O (usually 1-2µl of cell lysate or

mitochondrial preparation) before adding 200µl of Bradford reagent (BioRad) to either the lysate, or BSA standard curve samples. The samples were mixed well, then incubated for 5 minutes at room temperature before 200µl aliquots of each sample to a 96 well plate. The absorbance at 595nm of samples was measured on an ELx800 microplate reader (BioTek). The optical density was used in conjunction with the BSA standard curve to calculate the average protein concentration in the test samples.

2.5.4

SDS-PAGE

This method allows separation of denatured proteins according to their molecular weight. Casting and running of gels was performed using either the Hoefer Mighty SmallTM or Bio-Rad Mini-Protean® Tetra Cell system. Gels were cast with resolving phases of 12% or 14% polyacrylamide depending on the molecular weight of the proteins of interest (see Table 2.4 below). Addition of water above the resolving matrix allowed a smooth meniscus to form. This was removed after polymerisation was complete and a 3.75% stacking gel was added on top. Samples for SDS-PAGE analysis were incubated with sample dissociation buffer (final concentrations: 6.25mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, approximately 0.01% bromophenol blue and 100mM DTT) and incubated for 3 minutes at 95°C or 15 minutes at 37°C before loading. SDS- PAGE was performed in 1x running buffer (192mM glycine, 25mM Tris and 0.1% SDS) at 70V through the stacking gel and 150V though the resolving gel when using the Hoefer system, or a stable 200V when using the Bio-Rad system.

Table 2.4: Reagents and Volumes for SDS-PAGE. 12% resolving gel 14% resolving gel 3.75% stacking gel 30% acryl/bisacrylamide (29:1) 2ml 2.333ml 0.625ml 3.75M Tris/HCl pH 8.5 0.5ml 0.5ml -- 0.5M Tris/HCl pH 6.8 -- -- 1.25ml dH2O 2.395ml 2.062ml 3.02ml 10% SDS 50µl 50µl 50µl TEMED 5µl 5µl 5µl 10% APS 50µl 50µl 50µl Final Volume 5ml 5ml 5ml

2.5.5

C

S

General Coomassie Brilliant Blue (CBB) staining was used for visualising proteins after SDS-PAGE. Typically gels were incubated in CBB stain (45% methanol, 10% acetic acid and 0.2% Coomassie Blue R250) for 8 minutes at room temperature with agitation, before placing in de-staining solution (45% methanol and 10% acetic acid) for 10 minutes. The de-staining solution was then replaced with fresh de-staining solution and incubated at room temperature for a further 10 minutes or until the background of the gel was clear.

If greater sensitivity was required (as low as 5ng can be detected), InstantBlue (Expedeon, ISB1L) stain was used. Gels were washed briefly with water before staining with Instant Blue for at least 1 hour.

If mass spectrometry was to be performed on an excised band then the gel was washed 3 times for 5 minutes with dH2O and stained for at least 1 hour with SimplyBlueTM

SafeStain (Life Technologies, LC6060).

Images of Coomassie-stained gels that had been prepared by any of the methods outlined above were acquired using either an Epsom EU-35 scanner or the Bio-Rad ChemiDocTM _{MP system.}