Evolución de los instrumentos de protección

The next sections d escrib e th e egg white sep aratio n s carried out to examine th e effectiv en ess of th e deconvolution algorithm .

2.3.4.1 Sample preparation

The white of one hen egg was sep ara ted from its yolk and th e su p p o rtin g membranes were carefu lly removed. The egg white was th en diluted with 200 mL of 5 mM sodium phosphate b u ffer a t pH 6.0. This re su lte d in th e p recip itatio n of some components and so th e re s u lta n t solution was th en filtere d u sin g a B uchner funnel and Whatman No.l filte r paper. The solution was th en sto re d a t 277 K.

2.3.4.2 Separations

10 mL of diluted and filtered egg white were loaded onto th e Hiload 16/10 column p re-p a ck e d with Pharmacia S -S ep h aro se HP, which had been e q u ilib ra ted with 5 mM sodium phosphate b u ffer a t pH 6.0. The unbound

material was th en washed from th e column using fo u r column volumes of 5 mM sodium phosphate b u ffer, ie, 40 mL a t a flow ra te of 2 mL min Elution was carried out u sing a g rad ie n t of 0.2 M to 1.4 M sodium chloride in 5 mM sodium phosphate a t pH 6.0. The volume over which th e g rad ien t was carried o u t was v aried from te n column volumes to fo u r column volumes of elution bu ffer, to produce a v a rie ty of d ifferen t peak separations.

After elution had been c arried th e column was re g e n era ted with 1.4 M sodium chloride / 5 mM sodium phosphate b u ffer to remove all bound material before re-eq u ilib ratio n with 5mM sodium phosphate buffer. (Table 2.3 lists th e g rad ie n ts used in sep aratio n s c arried out.)

Fractions of fixed volumes of th e eluted material were collected and analysed by SDS polyacrylamide electro p h o resis (section 2.3.6).

Table 2.3 Gradients used in cation exchange separations Separation num ber sta rtin g volume mL finishing volume mL sta rtin g concentration of NaCl M finishing concentration of NaCl M 1 100.0 150.0 0.2 1.1 2 100.0 130.0 0.2 1.1 3 100.0 106.0 0.2 1.1 4 100.0 106.0 0.3 1.1 5 100.0 106.0 0.3 1.4 6 100.0 106.0 0.3 1.6

2.3.5 Analysis by sodium dodecyl sulphate-polyacrylamide gel

electrophoresis (SDS-PAGE)

A Bio-Rad Mini-PROTEAN II slab cell (Bio-Rad L aboratories Ltd.) v ertical slab electro p h o resis in stru m en t was used with an EPS 400/500 power u n it (Pharmacia Ltd.). D iscontinuous gels consisting a stack in g gel and a resolving gel were used in all gel analyses. The composition of th e gels

(Creighton, 1990) is shown in Table 2.4 and th e composition of th e ru n n in g b u ffer, staining and destaining solution is shown in Table 2.5. Table 2.4 Formulation of resolving and stacking gels

Component Resolving Gel

13.5% Stacking Gel 4% Acr y lamide/Bis ( 30% ) 13.5 mL 2.7 mL 1.5 M Tris-HCl pH 8.8 7.5 mL - 0.5 M Tris-HCl pH 6.8 - 5 mL 10% SDS 0.3 mL 0.2 mL 10% Ammonium p e rsu lp h ate 0.1 mL 0.1 mL TEMED^ 0.02 mL 0.02 mL Deionised water 8.6 mL 12 mL Total volume 30 mL 20 mL

1. TEMED; N, N, N’, N’- tetram ethylethylenediam ine

Table 2.5 Formulation of running buffer, staining and destaining solutions

5X Running b u ffer stock pH 8.3

IL solution: 15g Tris base 72g Glycine 5g SDS

Staining solution IL solution: 417 mL w ater 417 mL methanol 167 mL acetic acid Ig Coomassie Blue G-250 Destaining solution IL solution: 600 mL w ater 300 mL methanol 100 mL acetic acid 2.3.5.1 Sample Preparation

Fractions obtained from th e chrom atographic sep aratio n s were analysed using th e B radford protein assay (B radford, 1979) to determ ine which had th e la rg e st concentration of protein. When th is fractio n had been

identified a volume of th e fractio n containing betw een 200 jig and 300 pg of protein was tak en and a d ju ste d to

1 mL in an E ppendorf tu b e. This volume was used with all th e o th er fractio n s for consistency. 333 pi of 100%(w/v) trich lo ro acetic acid solution was th en added to each fractio n such th a t th e final concentration of trich lo ro acetic acid was a t least 25%(w/v). The fractio n s were then incubated a t 277 K for a t least 2 h ours. The p recip itated protein was th en obtained by cen trifu g atio n a t 13 000 rpm fo r 7 minutes. The su p e rn a ta n t was th e n carefully discard ed . 1 mL of acetone / 5 mM HCl solution was added to th e pellets of protein which were th en r e ­ suspended by vortexing.

The pellets were sp u n down as described above and again th e su p e rn a ta n t was discarded. 1 mL of acetone was th en mixed with each fraction. The pellets were recovered again as described above and dried in a speed vacuum desiccator (Savant, Speed vac sc 100). They were then dissolved in SDS-PAGE sample ru n n in g b u ffer (Table 2.6) and if n ecessary a d ju ste d to th e c o rre c t pH with 1-2 pL of Tris base solution. Before being loaded onto th e gels th e samples were boiled for 1 to 3 minutes to e n s u re th a t th e protein was completely d en atu red and spun a t 13 000 rpm to e n su re th a t any p articu lates were not loaded onto th e gels. Table 2.6 SDS-PAGE sample running buffer composition

Component Concentration Tris-HCl pH 6.8 62.5 mM Glycerol 10 % M ercaptoethanol 5 % or DTT 10 mM SDS 2.5 % Bromophenol Blue 0.05 % 96

2.3.5.2 Gel preparation

A 13.5% gel resolving gel was c ast using th e degassed solutions described in Table 2.4 above and allowed to polymerise for approxim ately 20 minutes (depending on th e ambient tem p eratu re) before th e 4% stacking gel was cast. This was allowed to polymerise. The wells were rin sed with ru n n in g b u ffer before being attach ed to th e ru n n in g a p p aratu s. Running b u ffer was th e n added to th e u p p e r and lower ta n k s and th e samples loaded into th e wells using a m icro-syringe.

A c u rr e n t of 20 mA per gel was applied for approxim ately 1 hour 15 m inutes or u n til th e dye fro n t reached th e bottom of th e gel.

On completion of th e electro p h o resis th e gels were removed from th e plates and th e stacking gels removed. The resolving gels were th en placed in stain in g solution (Table 2.5) for a t least 1 hour and destained with destain in g solution (Table 2.5). The destained gel was th en scanned to determ ine th e amounts of material in each band.