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If not required the gel apparatus was wrapped in Saran-wrap and stored at 4°C until use. All gels were poured with a thickness of 1.5mm.

Preparation o f samples for SDS-PAGE

Samples to be run on polyacrylamide gels were denatured by addition of an equal volume of 2x Laemmli gel loading buffer (4% (w/v) SDS, 0.02% (w/v) bromophenol blue, 20% (v/v) glycerol, lOOmM TrisHCl pH6.8 - all electrophoresis reagents from Sigma, Poole, UK) containing 200mM Dithiothreitol (Sigma, Poole, UK) and boiled at 100°c for 3 minutes. If the sample was too viscous to be easily loaded onto the gel (due to cell lysis releasing genomic DNA) it was sonicated for 5 seconds using a Soniprep 150 probe sonicator (MSB) at half power (corresponding to an amplitude of 18 microns on the sonicator). Denatured protein samples were stored at -20°C until required.

Loading and running polyacrylamide gels

Before loading, the combs in the gel were removed and the wells washed thoroughly with distilled water. The whole gel apparatus was then immersed into a tank containing fresh running buffer (25mM Tris, 250mM glycine, 0.1% SDS, pH8.3) and samples were loaded into the appropriate wells. 5 pi of pre-stained broad range molecular weight markers (Bio-Rad Laboratories, Hemel Hempstead, UK) were loaded into at least one lane of the gel as an aid to estimation of the molecular weights of proteins of interest as well as a visible marker of the efficiency of transfer to nitrocellulose. For 15-well and 10-well gels a maximum of 25pl and 40pi of sample was typically loaded respectively. The rest of the electrophoresis cell was assembled and voltage was applied using a power pack (Powerpac 300; Bio-Rad laboratories, Hemel Hempstead, UK). The samples were run through the stacking gel at a constant voltage of 40V and through the resolving gel at 80V. The applied voltage was

removed once the dye front had reached the bottom of the glass plates.

Transferring proteins to nitrocellulose

A freshly run gel containing separated proteins was removed from the glass plates and immersed in transfer buffer (48mM Tris, 39mM Glycine, 0.037% (w/v) SDS, 20% (w/v) Methanol, pH8.3) immediately after running. The gel formed the centre of a sandwich made up of nitrocellulose (Hybond ECL, Amersham-Pharmacia Biotech, Little Chalfont, UK), filter paper (Whatman Number 1) and fibre pads enclosed in a specialised cassette. The arrangement of the transfer sandwich is shown in Figure 2.7B. Once the transfer sandwich was assembled it was placed into the transfer apparatus and immersed into a reservoir containing transfer buffer. A constant current of 250mA was applied across the apparatus for 2 hours which was sufficient to transfer the majority of the proteins on the gel to the nitrocellulose, as judged by the transfer of the pre-stained molecular weight markers. The entire transfer system (i.e. the gel tank) was placed on ice during the transfer to ensure that minimal excess heat was generated by the electrical current, which can cause the formation of air bubbles between the gel and nitrocellulose. On some occasions transfer was done at 50mA for 12-16 hours at room temperature with equal quality of results. If the protein detection procedure was not done immediately after transfer, nitrocellulose sheets were kept at 4°C wrapped in Saran wrap moistened with PBS + 0.1% Tween.

Detection o f proteins on nitrocellulose using antibodies

The detection protocol was adapted from the instructions provided with the ECL Western blotting analysis system (Amershan-Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK).

Nitrocellulose membranes were incubated in 5% (w/v) Non-fat milk (Marvel, Premier brands, UK) in PBS + 0.1% Tween (PBS-T) for 1 hour on a shaking platform at room temperature. Membranes were then washed three times with PBS-T for a total of 20 minutes. The primary antibody was applied to the membranes at a typical dilution of 1:2000 in sterile filtered PBS-T containing 0.01% (w/v) sodium azide.

Sodium azide was added to ensure that antibody solutions did not become

contaminated. Membranes were incubated with primary antibody solution for 12-16 hours at 4°C on a shaking platform. After incubation with primary antibody,

membranes were washed three times with PBS-T for a total of 20 minutes. The secondary antibody ([horseradish peroxidase linked anti-mouse or anti-rabbit IgG antibody) was then applied at a 1:10,000 dilution in PBS-T to the membranes followed by a 1 hour incubation at room temperature on a shaking platform. Membranes were subsequently washed with PBS-T three times for a total of 20 minutes before reactive bands were visualised using ECL reagents and

chemiluminescence film (Hyperfilm ECL - Amersham-Pharmacia Biotech) according to the manufacturers instructions. It should be noted that due to the finite amounts of some of the channel subunit polyclonal antibodies available, antibody solutions were routinely reused 4-5 times with no observable loss in sensitivity. As stated above, the dilution of primary antibody used was typically 1:2 0 0 0, however on some occasions different dilutions were used where stated in figure legends. When the 9E10

hybridoma supernatant was used as the primary antibody, 50ml of culture supernatant was used. The supernatant was typically obtained from a sample of suspension culture and was centrifuged at 1300g to remove cells and cell debris.

A.

_Semi-dry

Direction o f transfer

Wet

B

Direction of transfer

H absorbent paper soaked in transfer buffer

G 3 nitrcKellulose

■■ negative electrcxle H positive electrode m fibre pads

Figure 2.7 Comparison o f semi-dr\ and wet electrophoretic transfer.

The semi-dr\ system (A) does not require immersion o f the w hole array in transfer buffer. The sheets o f absorbent paper are soaked in transfer buffer prior to assembly o f the sandwich and electrodes are placed directly onto the stack. This method gives even and rapid transfer. The wet system (B) requires assembly o f a sandwich, which additionally contains two fibre pads. Tire w hole sandwich is placed into a plastic cassette, which is in turn inserted into a electrode assembly with positive and negative faces. This whole

assembly is submerged into a tank containing transfer buffer and a constant current is applied. The conditions for wet transfer can vary , yet none can offer complete transfer o f all proteins to the nitrocellulose membrane. However, the mini gel

s\ stem used for our wet transfer yielded satisfactory results.