Protein techniques

2.7.1 Protein expression in oocytes

Oocytes were injected with 74 nl of mRNA at concentrations ranging 20-50 ng/μl and incubated at room temperature for 2 hours before transferring to 18°C overnight. If radio-labelling of proteins was carried out, the best 8 oocytes were selected after the 2 hour room temperature incubation. These oocytes were transferred to 100 μl Barth X containing 10μCi35S-methionine and incubated overnight at 18°C.

2.7.2 Protein extraction from oocytes

Oocytes were washed twice in Barth X before being homogenised in protein extract buffer (150 mM NaCl, 1% NP40, 50 mM Tris-Cl pH8) and 1 mM PMSF. Samples were recovered by centrifugation at 13 000 rpm for 5 minutes to remove cell debris and the middle layer removed. These samples were then spun down at 13 200 rpm for an additional 30 minutes. If performing radio-labelling the entire protein extract was mixed with 2X protein loading buffer at a 1:1 ratio. 20 μl of protein was taken from the supernatant and mixed with 20μl of protein loading buffer if Western blot analysis was to be performed. These extracts, either in loading buffer or protein extracted for immuno-precipitation (not in loading buffer) were stored at -80°C.

Protein samples denatured in protein loading buffer were heated for 3 minutes at 95°C and spun down for 5 minutes at 13 200 rpm before being loaded onto an SDS- PAGE gel. Samples were stacked through a 4% acrylamide stacking gel at 100V, followed by separation through a 10% acrylamide resolving gel in a tris-glycine

running buffer using the BioRad Mini Protean II system. Gels containing radio- labelled protein were dried for 1 hour at 80°C prior to autoradiography.

2.7.3In vitrotranslation

In vitro translation of 1 μl in vitro transcribed mRNA was carried out using the nuclease treated rabbit reticulocyte lysate according to the manufacturer’s instructions (Promega). Reaction products were radio-labelled using35S-methionine. Proteins were separated on a 10% acrylamide gel as described above.

2.7.4 Immuno-precipitation

Protein extracts were generated as explained in section 2.7.2. Extracts that were to be purified by immuno-precipitation were not denatured in protein loading buffer. Instead they were stored at -80°C. Protein extracts were defrosted on ice and at all times kept on ice. 200 μl protein extract was taken and placed in a new, already chilled, 1.5 ml Eppendorff tube. Primary antibody was added at the desired concentration. The extract with the primary antibody was mixed on a nutator (Eppendorff tubes on their side) at 4°C for 2 hours. Meanwhile beads (Protein G Sepharose used in this study) were swelled in protein extract buffer (150 mM NaCl, 1% NP40, 50 mM Tris-Cl pH8 and 1 mM PMSF). Typically 100 μl beads was swelled in 500 μl protein extract buffer. Beads were then washed 5 times in pre- chilled protein extract buffer, spun down at 5 000 rpm for 2 minutes each time. After final wash, 100 μl of beads were diluted into 500 μl protein extract buffer and stored at 4°C until required. For every 200μl sample, 50μl of swelled beads was added and mixed on a nutator at 4° for 2 hours. These mixes were then spun down at 5 000rpm for two minutes and washed 5 times in pre-chilled protein extract buffer. After the

final wash the beads were re-suspended in 30μl protein loading buffer, placed on ice for 1 hour and then stored at -80°C. (Note: Incubation of protein extract for more than 2 hours in primary antibody or sepharose beads increases non-specific binding and results in failed immuno-precipitation of target protein).

2.7.5 Western blotting

Protein extractions were carried out as in section 2.7.2. Typically 10% of an oocyte or 50% of an IP was separated on a 10% SDS-PAGE gel at 100 volts for 2.5 hours. In preparation for Western transfer, the Western transfer buffer was made up (20% Methanol, 39 mM Glycine, 48 mM Tris-HCl pH 7.5, 0.037% SDS) and placed at 4°C whilst the protein gel ran. After 2 hours of running the protein gel the PVDF membrane (Amersham), filter paper and fibre pads were prepared for Western transfer. The PVDF membrane was soaked in Methanol for 30 seconds to ensure activation, and then washed in distilled water. Filter paper, PVDF membrane and fibre pads were then equilibrated in a bath of Western transfer buffer at 4°C. After 2.5 hours the protein gel was stopped, transferred onto filter paper and then incubated at 4°C in Western transfer buffer for 30 minutes. Western blot sandwiches were then made (Fibre pad – Filter paper – Gel – PVDF membrane – Filter paper – Fibre pad). Sandwiches were locked in a cassette, correctly orientated and then placed in Western blot transfer apparatus (Biorad). Transfer of protein to the PVDF membrane was carried out at 4°C at 30 volts overnight (14 hours at least). PVDF membranes were isolated and blocked in 50% Marvel milk in 1X TBST (TBS + 0.1% Tween-20) for 2 hours at room temperature rolling. Membranes were washed 3x 10 minutes in TBST before addition of the primary antibody at specific concentration (anti-HA 1:200, anti-Myc 1:1000) in 5% Marvel milk and 1X TBST, which was left to bind

overnight, rolling at 4°C. Membranes with primary antibody bound were washed 3x 10 minutes in 1X TBST. Secondary antibody was added at desired concentration (HA westerns used secondary at 1:2000, Myc westerns at 1:10 000) in 5% Marvel milk and 1X TBST for two hours, rolling at room temperature. Membranes were then washed 3x 10 minutes in 1X TBST and the signal developed using EZ-ECL kit according to manufacturer’s instructions (Biological Industries).