Formas de cuantificación del MVC

The gel apparatus was dismantled. Gels were incubated in 50% (v/v) methanol for a m i n i m u m of 3 hours, then transferred to freshly prepared solution A (21ml of 0.36% (mass/v) NaOH and 1.4ml of 14.8M ammonium solution were combined and added dropwise wit h stirring to 0.8g of silver nitrate in 4ml of water, then the volume was m a d e up to 100ml with water) for 15 minutes. The gels were washed w ith copious volumes of deionised water then transferred to solution B (2.5ml 1% (mass/v) citric aci d and 0.25ml 38% (v/v) formaldehyde in 500ml of water) for 15 minutes or until protein bands were clearly visible. The gels were then fixed in 10% (v/v) glacial acetic acid, 40%

(v/v) methanol for 1 hour.

Autoradiography

The gel was fixed in 30% (v/v) methanol, 10% (v/v) glacial acetic acid for 1 hour at room temperature. The gel was transferred to an excess v o lume of A m p l i f y ™ for 15 minutes at room temperature. The gel w a s dried under vacuum at 65°C onto Whatman 3M M paper then e xposed to X-ray film for 4 hours to overnight at room temperature.

2.18.2 Detection of proteine on nitrocellulose

W^stern_bl<?fc

Proteins were transferred to nitrocellulose by a modification of the method of Towbin et a l . (1979). Gels were equilibrated in transfer buffer (25mM Tris-HCl, pH 8.3, 192mM glycine, 20% (v/v) methanol) for 15 minutes with gentle shaking. The gel was layered with an equal sized piece of nitrocellulose, then placed between 2 pieces of 3MM paper and the felt pads of the Biorad transblot apparatus. The apparatus was assembled and filled with transfer buffer. A 50 mA current was applied for 3 hours.

The apparatus was dismantled and the nitrocellulose was incubated in 50ml of TBST (50mM Tris-HCl, pH 7.5, 0.9% (mass/v) NaCl, 0.1% (v/v) Tween-20 containing 5% (mass/v) fat-free dried milk powder for 1 hour to block non-specific antibody binding sites. The nitrocellulose was transferred to 10ml block solution containing a 1:2000 dilution of rabbit anti-abrin C A- chain antibody and incubated at 37°C for 1-3 hours with gentle shaking. The filter was washed three times in 50ml TBST for 15 minutes.

Alkaline phosphatase development system

The ProtoBlot Alkaline Phosphatase System (Promega) was routinely u sed for Western blot analysis.

The washed filters were incubated in 15ml TBST containing 2/xl anti-(rabbit IgG)-alkaline-phosphatase conjugate for 1 hour at room temperature. The filters were washed three times for 15 minutes in 50ml TBST, then blotted dam p dry on a piece of 3MM paper. The damp filters were transferred to 10ml colour development substrate solution prepared by the addition of 66/il of 50mg/ml nitroblue tétrazolium (in DMF) and 33/il of 50mg/ml 5-bromo-4-chloro-3-indolyl phosphate (in DMF) to lOOmM Tris- HCl, p H 9 .5, lOOmM NaCl, 5mM MgC l 2 - When the colour had developed to the desired intensity, the reaction was stopped by

replacing the substrate solution with 20mM Tris-HCl, pH 8.0, 5mM EDTA. The filter was then air dried.

r12 5I1-protein A development system

After incubation with the primary antibody and washing, the filters were incubated in 10ml TBST containing lul [125I]- protein A (kindly provided by Dr P. Richardson, University of Warwick) overnight at room temperature, with g e n t l e shaking.

The filter was then washed 5 times for 5 min u t e s in 50ml TBST, then placed between plastic film. The f i l t e r was marked wit h IBI Glo-Juice, then exposed to X-ray film f o r 1 week.

2.19 PURIFICATION 0F 80L0BLE RECOMBIHART A-CHAIN 2.19.1 Solubility

Solubility of recombinant proteins was determined by centrifugation at I08,000g for 1 hour at 4°C. T h e supernatant containing soluble proteins was recovered, and t h e pellet was washed with lOOmM Tris-HCl, pH 8.5, 5mM EDTA, t h e n resuspended in an equal volume to the supernatant of lOOmM Tris-HCl, pH 8.5.

9-1» ? tmaoniurn sulphate precipitation

Concentration of recombinant abrin A-chain was performed using (NH4 )2S04 precipitation. Solid (NH4 )2S04 w a s added to the soluble fraction from cell sonicates (or periplasmic fractions) to 30% (mass/v) slowly at 4°C with stirring. T h e precipitated proteins were recovered by centrifugation at 15,000g for 15 minutes at 4°C, then resuspended in one tenth precipitation volume of lOOmM Tris-HCl, pH 8.5, 5mM EDTA. T h e sample was filtered through a 0.2 2/im filter.

The recombinant abrin A-chain was purified using an LKB DEAE-5PW UltraPac column (21.5X150mm) with a Beckman mo d e l 344 HPLC system. The column was equilibrated with the starting buffer (lOmM NaCl, 5mM sodium phosphate buffer, pH 7.5), and a 5ml volume of sample was applied. The column was w a s h e d with the starting buffer at 3ml/minute until the OD 280 rea c h e d the baseline level. The bound proteins were eluted with a gradient from lOmM to 500mM NaCl over 2 hours at 3ml/minute and eluate was collected in 3ml fractions. The fractions were a n a lysed by SDS-PAGE and Western blotting. Fractions containing A- c h a i n were pooled and dialysed against lOmM NaCl, 5mM sodium phosphate buffer, pH 7.5.

3.^9,4 Plv»t gn>h«rp»f CL-$P gfaromtqgrirhY

Recombinant A-chain was purified to homogeneity by chromatography on Blue Sepharose CL-6B. A 30Xlcm c o l u m n was packed with Blue Sepharose CL-6B resin, pre-swollen o v ernight in an excess of lOOmM NaCl, 5mM sodium phosphate buffer, pH7.5, at a flow rate of 0.275ml/minute. The column was equilibrated with starting buffer (lOOmM NaCl, 5mM sodium phosphate buffer, pH 7.5) and the baseline for OD 28o was established, at a flow rate of 0. 15ml/minute.

The A-chain sample was applied, and the column was washed at 0 . 15ml/minute with starting buffer until all non-bound materials had eluted, and the OD280 baseline was reached. A gradient to 500mM NaCl, 5mM sodium phosphate buffer, p H 7.5, was applied over 6 hours at 0. 15ml/minute and 0.5ml fractions were collected. The fractions were analysed for A-chain by SDS- PAGE and Western blotting. The fractions containing A -chain were pooled and dialysed against PBS.

2 . 2 0 A 8 8 A Y F O R I N V I T R O A C T I V I T Y O F R I P B

3.20.1 Sait-washing fiboaomeg

To remove bound elongation factors and aminoacyl-tRNAs, ribosomes were washed in 0.5M KC1 (Blobel, 1971). Ribosomes wer e incubated at a final concentration of 2mg/ml in 50mM Tris- H C 1 , pH 7.5, 0.5M KC1, 5mM MgCl2 for 1 hour on ice. The ribosomes were pelleted at 108,000g, at 2°C for 40 minutes. The supernatant was removed and the ribosomes were washed twice in 1 X TEP buffer (3.6mM Tris, 3mM NaH2P04 , 0.2mM EDTA). The washed ribosomes were resuspended at lnq/nl in 1 X TEP buffer.

in