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IjLil sample extract was added to 800/xl dH2 0 in an Eppendorf tube, and vortexed. 200/xl

BioRad protein assay reagent was added and mixed. The samples were transferred to plastic cuvettes (Elkay) and the absorbance at 595nm determined using a Shimadzu spectrophotometer. A standard curve was prepared using this method and known concentrations of bovine serum albumin (BSA). From this the concentration of protein in the samples was interpolated (Figure 2.3).

Absorbance (595nm) 0.4 0.35 0.3 0.25 0.2 0.15 O . I 0.05 0 y = 0.0668X + 0.0095 0 I 2 3 4 5 [BSA] (mg/ml)

Figure 2.3. Standard curves from bradford protein reagent using

DENATURED E. COLI EXTRACTS

1ml of an E. coli culture of known Agoo was centrifuged at MOOOrpm at room temperature for 1 minute. The cell pellet was resuspended in 100/xl 3x Laemmli buffer per 1 unit of absorbance at 600nm. This was boiled for 5 minutes, centrifuged and the supernatant retained. 10/xl of this extract was sufficient for visualising by Coomassie staining after SDS-PAGE.

DENATURED YEAST EXTRACTS

A known amount of cells (approximately 2x10*) was harvester and transferred to a 1.5 ml screw-capped tube. 50|xl Laemmli buffer was added and the cells lysed by five, 30s bursts at setting 5.0 in a Hybaid ribolyser. The beads and buffer were heated to 95°C for 5 minutes, then the beads were pelleted by centrifugation and all the buffer removed. This was used as the extract for determination of cellular copy number.

YEAST EXTRACTS - TCA

Denatured extracts were prepared using a trichloroacetic acid (TCA) precipitation method. Between 1x10* and 5x10* cells were harvested and transferred to a 1.5ml screw-capped Eppendorf tube. The pellet was resuspended in lOO/tl 20% TCA (prepared from 100% TCA solution - Sigma) and glass beads (0.4mm, BDH) added up to the meniscus. The cells were lysed in a Hybaid Ribolyser (setting 5.0 for 2x 20 seconds) at 4°C. The liquid was removed to a new tube and the beads washed with lOOjLtl 5% TCA which was added to the initial suspension. The suspension was centrifuged at 3000rpm for 10 minutes, a white protein pellet was formed. The supernatant was discarded and the pellet resuspended in 50/xl Ix Laemmli buffer. If the colour of the buffer at this stage was yellow, up to 25/xl IM Tris buffer pH 8.8 was

added until the blue colour was restored. The sample was boiled for 5 minutes, then centrifuged at 3000rpm for 10 minutes. The supernatant was retained as the extract. YEAST EXTRACTS - NATIVE

Native extracts were prepared from between 1x10* and 5x10* cells. 100/rl cold, 2x native lysis buffer and an equal volume of glass beads were added to the Eppendorf tube. The cells were lysed in a Ribolyser as above. A small hole was pierced in the base of each tube with a hot mounted needle. The tube was the placed inside a second Eppendorf tube, and the tubes spun in a swing out rotor (Beckman JS-6) at lOOOrpm for

kept in the upper tube, which was discarded. The lower tube was centrifuged at MOOOrpm for a further 10 minutes at 4°C and the supernatant retained as the extract. SDS PAGE PROTEIN GELS

Protein gels were run using either the BioRad mini protean system, for up to 15 samples or the Anachem SV2010 system for up to 36 samples. The table below gives recipes for common gels used (Table 2.3.1). Resolving gels were allowed to set for 30 minutes, with a layer of water on the surface to ensure a sharp interface between the resolving and stacking layers.

Gel% 6.5 7.5 8.5 10 Protogel 1083 1250 1417 1666 dH^O 3237 3070 2903 2654 3MTris pH 8.8 620 620 620 620 20% SDS 25 25 25 25 10% APS 25 25 25 25 Temed 6 6 6 6

Table 2.3.1. Preparation of SDS-PAGE gels (Resolving). All vo] giving a total of 5ml. Protogel (National Diagnostics) contains 3( 0.8% bis-acrylamide (37.5:1).10% APS was made weekly. The : requires 5ml/gel, the Anachem requires lOml/gel.

umes are in /xl, 3% acrylamide, BioRad system

Stacking gels were poured (Table 2.3.2) and the comb added immediately. All combs and plates were thoroughly washed before use in hot water and detergent, and handled only with gloves to reduce contamination by skin keratins. Samples of up to 25/xl were loaded, the gels were run at 200V for 45 minutes to 1 hour in Ix ORB. Molecular weight markers used were Sigma SDS-6H or SDS-7B, made up according to

manufacturers instructions or Novex Multimark, Sfil was loaded. Gels were either stained with Coomassie blue or transferred to nitrocellulose.

COOMASSIE STAINING OF GELS

For staining, the gel was transferred into a clean plastic box and Coomassie stain added to cover the gel. This was incubated at room temperature with agitation for 30 minutes. The stain was removed and kept for re-use. The stain was removed by three 30 minute

Protogel 330 dH20 1575 IM Tris pH 6 .8 313 20% SDS 12.5 10% APS 25 Temed 2.5

Table 2.3.2. Preparation of SDS-PAGE gels (stacking). All volumes are in fi\, sufficient for 1 BioRad gel, for Anachem twice as much is required.

washes in destain solution, with the inclusion of a sponge piece to help absorb the excess dye. After the stain was sufficiently removed the gel was soaked in 5% glycerol in dH2Û before air dying between 2 porous cellophane sheets (BioRad).

SILVER STAINING OF GELS

During this procedure all containers were either new or scrupulously cleaned with hot water and detergent. All solutions were made with freshly drawn water that was not stored in glassware and filtered through 0.2jLtm cellulose nitrate filters. The gel was handled as little as possible, and only with low protein, powder free gloves. The gel was soaked in 50% methanol, 12% acetic acid, 0.5ml/litre 37% formaldehyde (HCOH) in dH2 0 for 1 hour, followed by soaking in 50% ethanol in dH2 0 for 30 minutes. Both

soaks were at room temperature with agitation. The gel was then placed in 0.2g/litre sodium thiosulphate (Na2S203) in dH2 0 for 1 minute, followed by 3 washes in dH2 0 of 20 seconds each. Next, the gel was soaked in 2g/litre silver nitrate (AgNOg), 0.75ml/litre 37% HCOH in dH2 0 for 45 minutes at room temperature with agitation.

After 2 washes in dH2 0 of 30 seconds each the stain was developed in 60g/litre sodium

carbonate (Na2C0 3), 4mg/litre Na2S203, 0.5ml/litre 37% HCOH in (IH2O. The staining

was stopped by soaking the gel in 1% glacial acetic acid (CH3CO2H) in (IH2O just prior to reaching the required intensity. The gel was then air dried between porous cellophane.

SEMI-DRY TRANSFER

The SDS-PAGE gel was rinsed with transfer buffer then placed onto cut to size Hybond C-extra membrane (Amersham) which had been previously soaked in transfer buffer.

Schuell) soaked in transfer buffer and placed between the plates of a BioRad semi-dry transfer apparatus with the membrane closest to the anode. 13V was applied for 35 minutes. The efficiency of transfer was monitored by comparing the amount of prestained markers remaining in the gel to the amount transferred. The filter was soaked in Ponceau S solution for 5 minutes then rinsed in water. This allowed marking of the non-stained molecular weight standards in pencil, and also provided an approximate loading control. The stain was removed by washing in PBST.