Cálculos de Ingeniería

Radiolabelled proteins synthesized either by in vitro translation systems or by oocytes were generally analysed by one-dimensional SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Proteins of interest were often immunoprecipitated before analysis, but total translation products were also analysed. In some cases, where immunoprécipitation was not possible, proteins were analyed by two-dimensional

electrophoresis. Electrophoresis of proteins is discussed in detail in Hames and Rickwood (1981), and I give only a brief outline here.

XI.L.i One dipensiopal SDS-PAGE

Preparation of samples: Two different methods were used. In the first, immunoprecipitate pellets or l-10pl of in vitro translation mix or total oocyte homogenate were taken up in Sample Buffer 1 [0.2M Tris-HCl pH8.8, 2X(w/v) SDS, 5mM EDTA, 1M sucrose, 0.01% bromophenol blue, 3.3mM DTT], to a volume of 30jj1. Samples were heated to 100“C for 3 min, allowed to cool, then alkylated by the addition of iodoacetic acid to 70mM.

After incubation at room temperature for approximately 15 min, they were spun in a microfuge for 1 min before being loaded onto the gel. When the homogenate or translation mix contained 3asP-label led synthetic RNA, the samples were treated with RNase A (50pg/ml, room temperature, 10 min) before preparation.

In later experiments, a second method gave better resolution of proteins: IOjjI of homogenate was added to 10j-il of 2X Sample Buffer 2 [0. 125M Tris-HCl pH6.8, 4%(w/v) SDS, 20*(v/v) glycerol, 0.01% bromophenol blue, 5%(v/v)

2-mercaptoethanol]. Samples were heated to 100°C for 3 min and spun in a microfuge for 1 min before being loaded onto the gel. Immunoprecipitate pellets or samples of lpl translation mix were taken up in 20>j1 IX Sample Buffer 2, then treated in the same way.

In both cases half of each sample (corresponding to one eighth of an oocyte) was usually loaded, and the remainder stored at -20°C. When a further aliquot of the same sample was required, it was thawed, vortexed, spun for 1 min, and loaded onto the gel.

Markers: The markers used were a commercial preparation of ‘^C-labelled methylated proteins: lysozyme, 14.3kD; carbonic anhydrase, 30kD; ovalbumin, 46kD; BSA, 69kD; phosphorylase, 92.5kD; myosin, 200kD. 10pg of cytochrome c was also loaded onto each gel. This gave a visual indication of the resolution during electrophoresis.

Electrophoresis: Slab gels were run using a discontinuous buffer system based on the method of Laemmli (1970). Resolving gels were cast containing the appropriate percentage

acrylamide, 0.375M Tris-HCl pH8.8, 0.1% SDS, 0.04% ammonium persulphate, and 0.06% TEMED. A stock solution of 30%(w/v) acrylamide, 0.825% bisacrylamide was used. All the gels described in this thesis were 10% or 12.5% acrylamide. The stacking gel always had the same composition: 5%(w/v) acrylamide, 62.5mM Tris-HCl pH6.8, 0.1% SDS, 0.08% ammonium persulphate, 0.06% TEMED.

After loading of the samples, gels were usually electrophoresed at 20mA per gel until the dye had entered the resolving gel, when the current was increased to 30mA per gel. Occasionally, however, gels were run at a lower current for a

longer period. The electrophoresis buffer was 25mM Trizma, 0.192M glycine, 0.1% SDS.

Fixation and Fluorography: Gels were fixed for 1-48 h in 45%(v/v) methanol, 10%(v/v)'acetic acid. They were then fluorographed either by the method of Bonner and Laskey (1974) as described in Hames and Rickworth (1981), or by treatment with the commercial preparation ’En’Hance'. However, 'En3Hance' was used only with 10% acrylamide gels, since higher percentage gels tended to crack while drying down after this treatment. Dried, fluorographed gels were

autoradiographed by exposure to X-ray sensitive film at -70“C.

II.L.ii -TwQ-PimeDsiQaal Gel Electrophoresis

Two-dimensional analysis of proteins was carried out by a method based on that of 0 ’Farrell (1975).

Preparation of samples: Oocytes were usually homogenized in homogenization buffer (see II.M.ii), so that the same samples could be analysed on both one- and two-dimensional gels. For two-dimensional gels, one volume of homogenate was mixed with two volumes of lysis buffer [9.5M urea, 2%(v/v) NP-40, 5%(v/v) 2-mercaptoethanol, 1.6%(v/v) ampholines pH3.5-10, 3.4%(v/v) ampholines pH5-7]. Usually, 50>j1 were loaded per tube gel, corresponding to just under half an oocyte.

Isoelectric focusing: First dimension, isoelectric focusing gels 11cm long were cast in 13cm lengths of 2.5mm diameter glass tubing. The composition of the gels was: 4%(w/v) acrylamide (from a stock of 28.4% acrylamide, 1.6% bisacrylamide), 9.17M urea, 2%(v/v) NP-40, 1.7%(v/v) ampholines pH3.5-10, 3.3%(v/v) ampholines pH5-7, 0.012% ammonium persulphate and 0.07% TEMED. The gels were overlaid with 8M urea during polymerization, after which it was replaced with lOpl lysis buffer.

The gels were pre-run at 200V for 15 min, 300V for 30 min and 400V for 30 min, using 20mM NaOH as cathode buffer, and lOmM orthophosphoric acid as anode buffer. The lysis buffer and NaOH were then removed from the tops of the gels, and the samples loaded. They were overlaid with IOjjI of 9.17M urea, 0.083% ampholines pH 3.5-10, and 0.167% ampholines pH 5-7. The gels were then run at 400V for about 19 h.

After electrophoresis, the gels were removed from the tubing and transferred to equilibration buffer [70mM Tris-HCl pH6.8, 2.2%{v/v) SDS, 5.6X(v/v) 2-mercaptoethanol]. The gels were either loaded onto the second dimension after 30-60 min in equilibration buffer, or frozen at -70” C until second dimension gels could be run.

Determination of pH gradient: A 'blank' tube gel was always included when isoelectric focusing gels were run. After electrophoresis, this was removed from its glass tubing, cut into 1cm lengths, and each piece was incubated with 2ml distilled water for 20 min at room temperature. The pH gradient was then determined by measuring the pH of each solution.

SDS-polyacrylamide gel electrophoresis: For the second dimension, gel recipes were exactly the same as for

one-dimensional gels (see II.L.i). The resolving gel was first poured to a depth 2cm less than the height of the plates, then the stacking gel was poured to reach the top of the plates. Once this had set, the IEF gel was placed carefully onto it, and sealed into position with a melted mixture of IX agarose, 0.35% SDS, 70mM Tris-HCl pH6.8 and 0.01X bromophenol blue. The gels were usually run at 20mA through the stacking gel and 30mA through the resolving gel, but occasionally they were run at low current (5-10mA) overnight. They were then fixed and fluorographed as normal.