Section 2. 2 Methods 2.2.1 Cloning techniques
2.5 Protein analysis
Denaturing Polyacrylamide gel electrophoresis (SDS-PAGE). The discontinuous buffer gel system based on that of Laemmli (1970) was used for denaturing polyacrylamide gel electrophoresis (SDS-PAGE). Unless otherwise noted all gels were constructed with a 4 % polyacrylamide stacking gel and a 10 % polyacrylamide resolving gel. Typically electrophoresis of each gel was cairied out at a constant current of 15 - 20 mA throughout the stacking gel and 30 - 40 mA through the resolving gel.
Immunoprécipitation reactions. In vitro transcription/translation reactions were immunoprecipitated with either CAT or GUS antibodies as follows:
(a) Preparation of immunoprecipitin. Immunoprecipitin (Gibco-BRL), i.e. heat killed, formalin fixed Staphalococcus aureus cells, 1 g in 10 ml PBS, was centrifuged for 20 minutes at 3000 rpm and the supernatant discarded. The cell pellet was resuspended in PBS containing 10 % p-mercaptoethanol and 3 % SDS, and the sample incubated at 95 °C for 30 minutes. The cells were again centrifuged at 3000 rpm for 20 minutes, the supernatant discarded, and the cells resuspended in 900 |xl of NET/BS A .
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(b) Pre-clearing the sample. This removed proteins which would have bound non- specifically directly (i.e. not via the antibody) to the immunoprecipitin. 50 jiil of NET/BS A was added to 10 |il of coupled TnT reactions in 0.5 |xl microcentrifuge tube.
1.5 }xl of immunoprecipitin solution was added and incubated at room temperature for 15 minutes, then spun in a microcentrifuge at 13000 rpm for 2 minutes and the pellet discarded.
(c) Immunoprécipitation reaction. 5 jLil of the appropriate anti-serum was added to 50 jo,l of the supernatant of the precleared sample and incubated overnight at 4 °C. 6 pi of immunoprecipitin were added and incubated for 15 minutes at room temperature, spun in a microcentrifuge for 2 minutes at 13000 rpm, and the supernatant discarded. The pellet
was resuspended in 100 pi of NET/GEL/NaCl , spun in a microfuge for 2 minutes at 13000 rpm, and the supernatant discarded. The pellet was resuspended in 100 pi of NET/GEL/SDS , spun in a microfuge for 2 minutes at 13000 ipm, and the supernatant discarded. The pellet was resuspended in 100 pi of 10 mM tris.HCl (pH 7.5) and 0.1 % NP40, spun in a microfuge for 2 minutes at 13000 rpm, and the supernatant discarded. The pellet was resuspended in 1 x SDS-PAGE loading buffer containing 100 mM DTT, and the sample boiled for 2 minutes and spun in a microfuge for 2 minutes at 13000 rpm. The results were analysed by denaturing SDS-PAGE.
Western blotting. Proteins were separated by SDS-PAGE, before being transferred to PDVF membrane using a semi-dry transfer tank (Gibco-BRL).The membrane was left in blocking buffer (PBS, 20% (w/v) Marvel) either overnight at 4 °C or for 2 hours at 37°C with agitation. It was allowed to equilibrate to room temperature, and washed in washing buffer (PBS, 0.1 % (v/v) Tween 20, 1 % Marvel) for 10 minutes. The antiserum to be used was diluted in antibody diluent (PBS, 1 % (w/v) Marvel) to a dilution of 1:500 or 1:1000 (as stated in the figure legends), and incubated with the PDVF membrane for 30 minutes at room temperature, with agitation. The membrane was washed three times in washing buffer for 10 minutes each time, then incubated with a 1:1000 dilution of horseradish peroxidase labelled goat anti-rabbit IgG in antibody diluent for 45 minutes at room temperature. The PDVF membrane was then washed twice in 1 % Marvel in PBS for 5 minutes, then three times in PBS alone. The enhanced chemiluminescence (ECL) kit was used to develop the blotted membrane.