Chapter 2 Materials and Methods CHAPTER 2 Materials and Methods 2.1 Chemicals, reagents and kits Chemicals used in this work were of analytical grade or equivalent. All materials were handled and stored according to the manufacturers’ recommendations. The names of the suppliers of chemicals, reagents, enzymes and kits are given below: Table 2.1. List of the suppliers of chemicals, reagents and kits. Chemicals, Reagents and Kits Supplier Acetone (ALFA030699.K2) Acriflavine (01673) Acrylamide/ Bis solution (29:1), 40% (w/v) (1610146) Agarose (A7431) Ammonia (100112N) Ammonium ferric citrate (27882AC) Ammonium sulphate (100334C) Ammonium persulphate (007190438) Ammonium acetate (100134T) Anthrone (21451102) Avidin Horseradish Peroxidase Bovine Serum Albumin, fraction V (23209) Bromophenolblue Carboxymethylcellulose (2252596) Charcoal (33203) Citric acid (100813M) Concanavalin A (ConA) (L7647) Concanavalin A, fluorescein conjugate (ConA-fluorescein) (C827) Coomassie® Plus Protein Assay Reagent (1856210) Dimethylsulphoxide (DMSO) (103234) Disodium hydrogen orthophosphate (102494) D-glucose (101174Y) DNP (Dinitrophenylhydrazine) EDTA (disodium ethylenediamine tetraacetate) EnzChek Protease Assay Kit (E6638) Ethanol (28719) FM® 4-64 (T3166) Formaldehyde (F8775) VWR Sigma BioRad Sigma BDH BDH VWR BDH BDH BDH Molecular Probes Pierce Biotechnology BDH BDH VWR BDH Sigma Invitrogen Pierce Biotechnology VWR BDH BDH Sigma Sigma Invitrogen VWR Molecular probes Sigma 52 Chapter 2 Materials and Methods Fructose (103672G) BDH Glucose (G7528) Sigma Glass beads (G8772) Sigma Glycine (101193L) BDH Glycerol (1001184K) BDH HiTrap™ Phenyl Sepharose fast flow, low substitution column (17135301) Amersham Biotech Horseradish peroxidase Sigma Hydrochloric acid (103072L) VWR Isopropanol (7015975) BDH Luminol (5-amino-2,3-dihydro-1,4-phthalazinedione) Sigma Magnesium sulphate (291184P) VWR Micro Bio-Spin® Chromatography Columns (7326204EDU) BioRad MPB (N'-(3-maleimidylpropionyl) biocytin) Molecular Probes Methylene blue (340485C) VWR Peptide Institute MOCAc-Ala-Pro-Lys-Phe-Phe-Arg-Leu-Lys(Dnp)-NH2 Inc.,Osaka, Japan. Nile red (N3013) Sigma Ninhydrin (25905107) BDH PBN CSIR South Africa Peptone (P/1160/50) Fisher Chemicals Periodic acid (P0430) Sigma Potassium disulphite (P2522) Sigma Potassium phosphate (P5379) Sigma Proteinase A (P8892) Sigma Protein marker, broad range (P7702S). BioRad Propidium iodide (PI) (P4170) Sigma Ribonuclease A (RNAse A) (R5500) Sigma Silver nitrate (102332Y) BDH Silcorel ® AFP10 (63280) VWR Sodium acetate (102363P) BDH Sodium chloride (102414J) BDH Sodium dihydrogen orthophosphate (307164T) BDH Sodium citrate dihydrate (S4641) Sigma Sodium hydroxide (102524X) BDH Sodium metabisulphite Sigma Sodium sulphate (100073A) BDH Sucrose BDH Sulphuric acid (102760B) BDH TEMED (1610801) BioRad Wheat Germ Agglutinin, fluorescein conjugate (WGA-fluorescein) (W834) Invitrogen Yeast extract (LP0021) Oxoid Yeast nitrogen base (239210) BD Difco 53 Chapter 2 Materials and Methods 2.2 Agar slopes, media, buffers and solutions PYN (Peptone Yeast Extract Nutrient) agar slopes Bacteriological peptone 0.35 g, 0.30 g yeast extract, 0.20 g KH2PO4, 0.10 g (NH4)2SO4, 0.10 g MgSO4*7H2O, 10.0 g glucose and 2.0 g agar were added to 100 mL dH2O and autoclaved at 121°C for 15 min. The autoclaved medium was cooled to 50°C and the agar solution (5 mL) was poured into sterile screw capped glass vials. The agar slopes were allowed to dry and stored at 4°C. YPD (Yeast Extract Peptone Dextrose) agar slopes Glucose (20.0 g) was added to dH2O (10 mL) and autoclaved at 121°C for 15 min. Peptone (20.0 g), yeast extract (10.0 g) and agar (20.0 g) was added to dH2O (90 mL) and autoclaved at 121°C for 15 min. The autoclaved glucose solution was added to the autoclaved nutrient medium. The medium was cooled to 50°C and the agar solution (5 mL) was poured into sterile screw-capped glass vials. The agar slopes were allowed to dry and stored at 4°C. PYN medium The liquid medium (1,000 mL) was the same formulation as the agar slopes but without agar addition. YPD medium The liquid medium (1,000 mL) was the same formulation as the agar slopes but without agar addition. Peptone, yeast extract and glucose were autoclaved together. YNB (Yeast Nitrogen Base) medium Yeast nitrogen base (1.80 g) and (NH4)2SO4 (4.91 g ) was added to 250 mL dH2O and autoclaved at 121°C for 15 min. PBS (Phosphate Buffered Saline) NaCl (40.0 g), KCl (1.0 g), Na2HPO4 x 2H2O (7.2 g) and KH2PO4 (1.0 g) was dissolved in 4.9 L dH2O. The pH was adjusted to 7.2 with 1N HCl, filled to 5 L with dH2O and the buffer was stored at 4°C. 54 Chapter 2 Materials and Methods TRIS (Tris(hydroxymethyl)aminomethane) buffer C4H11NO3 (24.23 g) was dissolved in 990 mL dH2O. The pH was adjusted to 7.2. The volume was filled to 1 L with dH2O and stored at 4°C. 2.3 Yeast strains The yeast strains employed in this study were industrial lager and ale strains and a yeast GFP clone of S. cerevisiae. The yeast strains were identified by growing them on peptone-yeast extract glucose nutrient agar plates for 48 h at 25C and 37C (Figure 2.1). Lager strains grew at 25C and not at 37C, whereas ale strains grew at both temperatures (Donhauser, 1995; Stewart and Russell, 1998). The Yeast GFP (Green Fluorescent Protein) Clone YPL154C (Invitrogen, Paisley, UK), used in this study, is part of the Yeast GFP Clone Collection which is a S. cerevisiae yeast strain collection expressing full-length Open Reading Frames (ORFs) containing a GFP (S56T) tag at the carboxy terminal end. The Yeast GFP Clone was supplied as a stab culture in LB (Luria Bertani) agar. To prepare a glycerol stock culture for long-term storage, a sterile loop was used to inoculate a colony from the stab culture in 5 mL YPD medium and this was incubated at 30°C overnight at 150 rpm. Sterile 80% glycerol (0.9 mL) was added and mixed thoroughly. This stock culture was dispensed into sterile Eppendorf tubes and frozen at –80°C. The Yeast GFP Clone was revived by transferring a small portion of the frozen sample onto a YPD agar slope, and incubating at 25C until sufficient yeast colonies had formed. Subsequently the agar slopes were stored at 4°C until needed. The industrial strains were maintained on YPD agar slopes at 4°C. 55 Chapter 2 Materials and Methods Ale yeast strain 25C 37C Figure 2.1. Growth of lager and ale yeast strains on peptone-yeast extract glucose nutrient agar plates for 48 h at 25C and 37C. S. cerevisiae GFP (Green Fluorescent Protein) clone YPL154C The Yeast GFP (Green Fluorescent Protein) Clone YPL154C used in this study is part of the Yeast GFP Clone Collection. The Yeast GFP Clone Collection is a S. cerevisiae Lager yeast yeast strain collection expressing full-length strainOpen Reading Frames (ORFs) containing a GFP (S56T) tag (Tsien, 1998) at the carboxy terminal end. The clones with the conventional GFP moiety (N- or C-terminal) are able to express chimeric fluorescent proteins that can be visualised by fluorescence microscopy at 488 nm (Niedenthal et al., 1996). The GFP fusion proteins are integrated into the yeast chromosome through oligonucleotide-directed homologous recombination and are expressed using endogenous promoters. The Yeast GFP Clone Collection of S. cerevisiae tagged ORFs was generated by Dr Erin O’Shea and Dr. Jonathan Weissman at the University of California, San Francisco (Huh et al., 2003). The Yeast GFP Clone YPL154C is a S. cerevisiae strain containing the GFP tagged ORF at the chromosomal location of the PEP4 gene (systematic name: YPL154C). 56 Chapter 2 Materials and Methods The PEP4 gene encodes proteinase A (EC 3.4.23.4). Proteinase A is a proteolytic enzyme that belongs to the vacuolar aspartic proteinase family. It is synthesised as a zymogen and is required for the posttranslational precursor maturation of vacuolar proteinases. Proteinase A has a molecular weight of about 42 kDa. The primary structure of the mature protein has been determined by amino acid sequencing (Dreyer et al., 1986). It has N-linked carbohydrates attached at two positions, and has an active site characteristic of aspartic proteases. The amino acid composition includes 43% polar residues and 12% aromatic amino acids. Proteinase A is a glycoprotein containing 7.5% (w/w) mannose and 1% (w/w) glucosamine and galactosamine and has an isolectric point of 4.4 (Meussdoerffer et al., 1980). Pr A is the most important protease in beer or in fermenting wort because it has the highest activity for degrading foam-active proteins at pH 4.0-4.5 (Kondo et al., 1998; Kogin et al., 1999). The mechanism of proteinase A excretion from yeast cells under fermentation conditions has never been investigated in detail (Kondo et al., 1998). The genotype of the parent haploid S. cerevisiae strain (ATCC 201388) is: MATa his31 leu20 met150 ura30. The strategy used to generate the Yeast GFP Clone Collection is described below (Figure 2.2). PCR (Polymerase Chain Reaction) products containing the GFP tag at the Cterminus and a selectable marker gene were generated for each ORF. PCR products were transformed into a haploid parent yeast strain to generate a C-terminally GFP tagged fusion protein for each ORF through homologous recombination. The result was a fusion protein containing a GFP at the C-terminus (Huh et al., 2003). The GFP was excited with the 488 laser line of an argon-ion laser and the fluorescent emission was collected at a wavelength of λ = 498–533 nm. 57 Chapter 2 Materials and Methods PCR product Homologous recombination Chromosome Fusion Protein Figure 2.2. Strategy for library construction of yeast GFP clone YPL154C (Huh et al., 2003). 2.4 Determination of yeast viability Yeast viability was assessed using methylene blue staining and an improved Neubauer haemocytometer (EBC Analytica Method 2.2.2.3). In addition, yeast viability was assessed using fluorescence staining with fluoresceindiacetate (FDA, 0.5 g FDA in 100 mL ice cold acetone) and propidium iodide (PI, 7 mg in 100 mL TRIS-buffer, pH 7.5). Viable cells contain esterases in their cytoplasm which cleave FDA. The released fluorescein emits green fluorescence after excitation at 488 nm. Dead cells do not contain esterases which cleave FDA but PI can enter through the damaged cell membrane and intercalates with the DNA in the nucleus. After excitation at 488 nm these cells show red fluorescence (see Figure 2.3). Five hundred cells were counted for each measurement. 58 Chapter 2 Materials and Methods Figure 2.3. Viability determination of yeast cells using fluorescence microscopy. Cells exhibiting green fluorescence are viable; cells exhibiting red fluorescence are non viable. 2.5 Beer production in the ICBD pilot plant All malt worts were produced in the ICBD 2 hL pilot brewery. The malt (Optic) was provided by Pure Malt (Haddington, UK) and was stored at a constant temperature of 11C prior to use. The high gravity wort was brewed at specific gravity 1.080 (20 Plato) and the lower gravity wort with a specific gravity of 1.048 (12 Plato). The malt was milled with a four hammer ‘Essex Major’ mill (Christy-Hunt, UK) to produce the fine grist necessary for the mash filter. The malt was mashed in with a liquor/grist ratio of 2.15:1 for high gravity worts and 2.5:1 for lower gravity worts. The mashing temperature was maintained at 65C for 1 h and the temperature raised to 74C for mashing off. Wort separation was achieved by employing a Meura 2001 pilot mash filter (Meura, Belgium). The wort was collected in a combined kettle/whirlpool (Briggs, UK) and boiled for 1 h at an evaporation rate of approximately 10% per h. During wort boiling, the worts were hopped with kettle pellets to obtain beer bitterness of 16 IBU after dilution to an alcohol concentration of 4.5% (v/v). The water used for dilution was brewing liquor purged with CO2 to minimise oxygen levels in the diluted beer. Fermentation was carried out in Briggs designed and manufactured cylindro-conical fermenters (Burton-upon-Trent, UK) with a total volume of 340 L. Worts at gravities of 12 Plato and 20 Plato were fermented using either lager or ale strains as specified in the experimental section. The wort dissolved oxygen content was adjusted to 1 mg/Plato and the pitching rate was approximately 106 cells/mL/Plato for both lager and ale fermentations. The fermentation temperature was maintained constant at 12C for lager fermentations and 20C for ale fermentations. The conditioning temperature was 5C for lagers and 9C for ales and in both cases the temperature was adjusted to -1.5C two days prior to filtration. Conditioning duration was approximately 5-7 days for low gravity fermentations and 10-12 days for high gravity fermentations. 2.6 Beer production at the Foster’s Brewery in Yatala, Brisbane, Australia 59 Chapter 2 Materials and Methods Worts at different gravities were produced in the Foster’s brewery, Yatala, Brisbane, Australia using maltose syrup adjunct (Table 2.2). In case of the 10 Plato and 14 Plato brews, the worts were hopped after filtration using iso-alpha extracts. The 18 Plato brew was hopped with kettle pellets during wort boiling to obtain a wort bitterness of 56 IBU after wort boiling. For an overview of the main brewing parameters for the three beer brands produced at the Foster’s brewery see also Table 2.3. Table 2.2. Malt to adjunct ratio of worts produced at the Foster’s brewery. Wort gravity 10 Plato 14 Plato 18 Plato Malt (%) 100 67 80 Adjunct (%) 0 33 20 Fermentation was carried out in cylindro-conical fermenters with a capacity of 5,000 hL. Worts (10 Plato, 14 Plato and 18 Plato) were fermented using S. cerevisiae (lager type) yeast strains. The wort dissolved oxygen content was adjusted to 8 mg/L for the 10 Plato and 14 Plato brews and to 14 mg/L for the 18 Plato brew. The pitching rate was 106 cells/mL/Plato for the 10 Plato and 14 Plato brews. The 18 Plato brew was pitched with 18.5 x 106 cells/mL. The maximum fermentation temperature was 18C for the 10 Plato and 14 Plato fermentations and 20C for the 18 Plato fermentation. Conditioning temperature was maintained at 5C and the temperature adjusted to -1.5C one day prior to filtration. Table 2.3. Characteristics of the three main beer brands produced in the Foster’s brewery. PARAMETER BRAND A BRAND B BRAND C Wort gravity 10 Plato 14 Plato 18 Plato Expected dilution ratio 1.25 1.25 1.90 Malt/adjunct ratio Medium Medium Plus High Dilution ratio 1.25 1.25 1.90 Roasted barley No No Yes Kettle hopped No No Yes Wort cooling temp 10C 10C 14C Lager yeast strain Strain A Strain A Strain A 60 Chapter 2 Materials and Methods Fermentation temp 18.5C 18.5C 20.0C Silica Gel / PVPP No Yes No Papain used Yes No Yes Tetra hops No No Yes Reclaim allowed Yes No Yes 4.90 3.50 Sales Alcohol (v/v) 2.7 Specific gravity and pH Wort (3 mL) was adjusted to 20C and centrifuged with a DuPont Sorvall RC Refrigerated Centrifuge (Sorvall UK) at 2,800 g for 10 min. The sample was degassed before the specific gravity was measured using a PAAR Model DMA 46 Digital Density Meter (Paar Scientific, UK). The specific gravity of wort at 20C was expressed as the density relative to water at a temperature of 20C (SL 20/20). In technical terms, the sugar content of wort is typically expressed as extract in degrees Plato. One degree Plato corresponds to a 1% (w/w) sugar solution of sucrose. Therefore, wort with 1 Plato has the same density as a 1% (w/w) sugar solution of sucrose at 20C relative to distilled water at 20C. The pH was measured using a Hanna Instrument 9321 Microprocessor pH meter (Hanna Instruments, USA) after calibration at pH 7 and pH 4. 2.8 Total protein concentration Total protein content was determined using the Bradford protein assay (Bradford, 1976; Dale and Young, 1987; Dale et al., 1989). Coomassie® Plus Protein Assay Reagent (1mL) (Pierce Biotechnology) was added to the sample (0.5 mL) and thoroughly mixed. The mixture was allowed to stand for 5 min and the absorbance measured using a Philips PU8700 UV/visible spectrophotometer at a wavelength of 595 nm. The concentration of total protein was calculated using Bovine Serum Albumin (BSA), fraction V (Pierce Biotechnology) as a standard. 2.9 Free alpha-amino nitrogen (FAN) in wort by colorimetry Wort samples (5 mL) were collected throughout fermentation and maturation, centrifuged at 2,800 g for 10 min and the supernatants analysed for free amino nitrogen (FAN) using the EBC standard method (EBC Analytica, 2005). The method gives an 61 Chapter 2 Materials and Methods estimate of amino acids, ammonia and, in addition, the terminal alpha-amino nitrogen groups of peptides and proteins. The principle of the method is that ninhydrin is reduced in the presence of alpha-amino groups at pH 6 to 7. The reaction between reduced ninhydrin and non-reduced ninhydrin results in a coloured product which can be measured at 570 nm. Wort samples were diluted to a concentration of 1-3 mg alpha-amino nitrogen/L. The diluted sample (2 mL) was transferred to a test tube and colour reagent (1 mL) (Na2HPO4 x 12 H2O (100 g), KH2PO4 (60 g), ninhydrin (5 g), fructose (3 g) in distilled water (1 L) added. The mixture was placed in a boiling water bath for exactly 16 min and cooled in a water bath at 20C for 20 min. Five mL dilution solution (KIO3 (2 g) in 600 mL dH2O and 400 mL ethanol 96% (v/v)) was added and the absorbance measured against a blank (2 mL water instead of the sample). Glycine solution (0.1072 g in 100 mL dH2O) was used as a standard. The glycine solution was diluted 100 fold so that the diluted solution contained 1 mg alpha-amino nitrogen/100 mL. With each set of alpha-amino nitrogen determinations, three replicate glycine standards were used (2 mL glycine standard instead of the sample) and the absorbance measured against the blank. The free alpha-amino nitrogen was calculated using the following formula: FAN (mg/L) = (A1 / A2) x 2 x d A1: Absorbance of the sample at a wavelength of 570 nm in 10 mm cells A2: Absorbance of glycine standard solution at a wavelength of 570 nm in 10 mm cuvettes d: dilution factor 2.10 Amino acid spectra of wort and beer by High Performance Liquid Chromatography (HPLC) Separation of amino acids was achieved by gradient elution, high performance liquid chromatography, using fluorescence as a means of detection (Hoff et al., 1978; Mackey and Beck, 1982). 2.11 Total polyphenols in wort and beer Samples to be analysed were centrifuged at 2,800 g for 10 min and the supernatants were analysed for total polyphenols using EBC methodology (EBC Analytica, 2005). The principle of the method is that polyphenols react with ferric iron in alkaline solution and the resulting red colour is estimated at 600 nm. The sample (10 mL) and CMC/EDTA (8 mL) (carboxymethylcellulose (10 g), disodium ethylenediamine tetraacetate (2 g) in 1 L 62 Chapter 2 Materials and Methods dH2O) were transferred to a 25 mL graduated flask with ground glass stopper and mixed well. Ferric reagent (0.5 mL) (3.5% (w/v) green ammonium ferric citrate (16% (w/v) Fe) was added and mixed well. Ammonia reagent (0.5 mL) (concentrated ammonia diluted with 2 volumes dH2O) was added to the mixture and distilled water added to a total volume of 25 mL. The blank was treated similarly, but instead of the ferric solution water was added. The solutions were allowed to stand for 1 min before measuring the absorbance of the sample against the blank at a wavelength of 600 nm. The concentration of total polyphenols (mg/L) was determined by: A600 x 820. 2.12 Determination of pentose sugars with phloroglucinol The five carbon sugar content of the samples was measured using an adapted version of the method of Douglas (1981). The reagent was prepared by combining 110 mL acetic acid, 2mL concentrated hydrochloric acid, 5 mL 20% phloroglucinol (1 g in 5 mL ethanol) and 1 mL 1.75% glucose (1.75 g/100 mL). A standard xylose solution (100 mg/100 mL) was prepared and diluted 1:10 (10 mg/100 mL). From this solution, standards with xylose concentrations of 7.5, 5.0 and 2.5 mg/100 mL were prepared and a standard curve created. This standard curve was used to calculate the concentration of pentose sugars in the samples. 2.13 Determination of pentose sugars in freeze dried proteinaceous material Approximately 4 mg of sample was dissolved in 2 mL water, the exact weight being noted, 500 µL of each standard was placed in three test tubes and 500 µL of each sample was placed in three test tubes and 500 µL of water was placed in one tube as blank. To each tube 2,500 µL reagent was added and mixed by vortexing. The tubes were placed in a boiling water bath for 25 min with marbles on top of each tube to prevent evaporation. Positive pentose samples turn pink and negative samples yellow. The tubes were cooled in a basin of cold water. The absorbance of the standard and the samples was measured at 552 nm and 510 nm against the water blank. The absorbance value at 510 nm was subtracted from the absorbance at 552 nm. The resulting value was used to calculate the weight of pentose sugar using the equation generated with the standard curve. This gave the amount of pentoses in 100 mL protein solution. Subtracting the 510 nm absorbance value from the 552 nm value removes interference from non-pentose sugars. This assay was standardised on xylose but also detects arabinose and ribose to the same extent. 2.14 Determination of pentose sugars with phloroglucinol in wort and beer 63 Chapter 2 Materials and Methods The five carbon sugar carbohydrate content of the samples was measured using the method of Douglas (1981) as discussed above (2.12). 2.15 Total carbohydrates in wort and beer by spectrophotometry The six carbon sugar content was measured using the EBC method 6.26 (Analytica EBC, 1995). The principle of this method is that if carbohydrates are subjected to acid catalysed dehydration, furfural derivatives are formed that react with anthrone (9-dihydro10-oxoanthracene) giving a blue/green compound (Yadev et al., 1969). The assay was standardised on glucose, but was found to detect 90% ((w/w) of total sugar present) of fructose, 60% galactose, 36% rhamnose and 51% mannose. The assay does not measure five carbon sugars. 2.16 Total carbohydrate determination in freeze dried proteinaceous material The six carbon sugar carbohydrate content of the various extracted glycoproteins was measured using an adapted version of the EBC method 6.26 (Analytica EBC, 1995). The reagent was prepared by placing 170 mL sulphuric acid in a 200 mL glass measuring cylinder, this was made up to 200 mL with water and mixed with a glass rod. The hot mixture was cooled by placing in a beaker and running cold tap water through the beaker. Anthrone (200mg) was placed in a 200 mL volumetric flask and filled with the sulphuric acid. The flask was refrigerated at 4ºC and the volume made up to 200 mL when required. Approximately 5 mg of sample was weighed and the exact amount noted and then placed in 10 mL water and diluted as required. A glucose standard (400 mg/L) was prepared and diluted 1:10 on the day of use. The standard (750 μL) was placed in three glass test tubes and 750 μL of the diluted sample was placed in three test tubes. Water (750 μL) was placed in one tube as a blank. The anthrone reagent (2,500 μL) was added to each tube and mixed by vortexing. The tubes were placed in a water bath at 95 ºC for 20 min with marbles on top to minimise evaporation. The tubes were cooled in a basin of cold water and re-mixed by vortexing. The absorbance of the standard and the samples was measured at 625 nm against the water blank using glass cuvettes. 2.17 Determination of glucose, D-fructose, sucrose, maltose and maltotriose by HPLC Separation of carbohydrates was achieved by high performance anion exchange (HPAE) (Analytica EBC, 1995). At high pH, carbohydrates are partially ionised and can therefore be separated by anion exchange. Detection was continuous by a pulsed amperometric detector (PAD) and involved measuring the electrical current generated by 64 Chapter 2 Materials and Methods the oxidation of carbohydrates by the high pH of the eluent at the surface of a gold electrode. Instrumentation used was a Dionex PAD (Pulsed Electrochemical Detector) with gold electrode, a Gilson 302 pump, a Gilson 305 pump, a Gilson 802 Manometric Module, a Gilson 811B Dynamic mixer, a Hewlett Packard 1050 auto injector, a Dionex eluent degas module and a Hewlett Packard Chemstation data handling (HP3365) system. Columns for separation were a Dionex Carbopac PA-100 Guard column, 4 x 50 mm and a Dionex Carbopac PA-100 column, 4 x 250 mm. Wort samples were diluted according to their specific gravity (Table 2.4). Internal standard (180 μL) was then added to diluted sample (900 μL) and mixed well. Samples were prepared in 1.5 mL glass vials and then capped. Table 2.4. Dilution of wort samples for carbohydrate determination by HPLC. Wort gravity Dilution Factor 1.050- 1.046 1:200 1.031- 1.034 1:100 1.018- 1.011 1:50 2.18 Determination of ethanol concentration by GC Ethanol concentrations were determined using a Chrompack CP 9000 gas chromatograph (GC) (Chrompack International BV, Middleburg, The Netherlands) with a packed column (10% silicone OV-1 on Chromsorb WHP, 80-100 mesh), splitless injector and flame ionisation detector (FID), and a Hitachi-Merck D2000 integrator (Baird & Tatlock, Essex, England). The column was a Chrompack CP SIL 5CB, 10 m x 0.32 mm (Chrompack International). Butanol was used as an internal standard. 2.19 Determination of ethanol concentration by distillation This analysis was carried out according to the Institute of Brewing Recommended Methods of Analysis (1997). At the end of fermentation, 200 mL of liquid was filtered through a Whatman No.2 filter paper. The gravity of the filtrate was measured to give the ‘Final Gravity’. Filtrate (100mL) was placed in a 1 L round bottomed flask with some water and distilled using a glass original gravity still and a Bunsen burner. The condensate was collected from the condenser into a 100 mL volumetric flask and the distillation conducted until 85 mL of spirit (known as Spirit Indicator) had been collected. The Bunsen was then removed and the still allowed to cool. The contents of the round flask, (the “residue”), was 65 Chapter 2 Materials and Methods made up to 100 mL with water and the same procedure carried out with the spirit indicator. The gravity of the residue was measured giving the ‘Residual Gravity’. The gravity of the Spirit Indicator was taken and multiplied by 997.15 to convert the value to density. The density figure was read off an alcohol table to give the % ethanol concentration by volume. 2.20 Determination of foam stability Beer foam stability measurements according to the Nibem principle are based on the determination of the time during which the foam-collar descends 10 and 30 mm in a standard beer glass (Van Akkeren, 1998). The moment the foam has descended to 10mm under the lip of the glass, a timer was activated. The time course over the next 30 mm was measured digitally. A movable system of electrodes, which responds to the conductivity of the foam, measures the foam level. A long needle is situated in the middle of the electrode surrounded by four shorter needles. When the foam is touched by one of the four shorter needles, contact is made between the long needle in the middle and one of the shorter needles. The descending movement of the electrodes stops when the contact between the needles is disrupted due to the collapse of the foam. Foam was generated in a glass by running beer directly from the bottle through a foam flasher unit. A pressure of 0.6 bar was applied to the head space of the bottle, using ultrapure CO2, which forced the beer out of the bottle through the foam flasher unit into the standard glass thus generating foam. The glass was then placed into the Nibem apparatus (Haffmans/Holland) and the Nibem value determined. The Nibem value quoted in this study is the time (in sec) for the foam to collapse over 30 mm. Each sample was analysed in triplicate and the mean reported. 2.21 Hydrophobic polypeptide analysis Samples to be analysed (5 mL) were centrifuged at 2,800 g for 10 min and the supernatant analysed for hydrophobic polypeptide content using the method developed at Brewing Research International (Bamforth, 1995). Sample temperature was adjusted to 20C and diluted 20-fold before being analysed for hydrophobic polypeptide content. The method employed a 1mL HiTrapTM Phenyl Sepharose fast flow low substitution column (Amersham Pharmacia Biotech). The principle of this column is that substances are separated on the basis of their varying strengths of hydrophobic interactions with hydrophobic ligands immobilised to an uncharged phenyl sepharose matrix. In the experimental procedure for the measurement of foam-positive polypeptides, diluted samples (3 mL) are passed through to the HiTrapTM column of which the first 1.5 mL is discarded. The second 1.5 mL of the sample is collected as eluate for the assessment 66 Chapter 2 Materials and Methods of the total protein concentration (Materials and Methods Section 2.8). The hydrophobic polypeptide content is calculated as follows. The diluted sample is passed through the HiTrapTM column and the total protein concentration measured before (A) and after the column (B). Reading (B) represents that protein that does not bind to the column and which therefore is characterised as hydrophilic. The subtraction of (B) from (A) is equal to the amount of hydrophobic protein, which binds to the column (Figure 2.4). Each sample was analysed in triplicate and the mean was calculated for every sample that was analysed using this technique. Diluted wort sample (A) Hi TrapTM Column y y y y y y y y y y Phenyl-Sepharose yx xy yx xy yx xy yx xy yx xy Hydrophobic Protein Eluate (B) Hydrophobic Protein Content = A-B Figure 2.4. Method for the analysis of hydrophobic polypeptides (Bamforth, 1995). 2.22 Measurement of extra-cellular proteinase A activity The assay procedure (Kondo et al., 1998) involved using the highly fluorescent (7methoxycoumarin-4-yl)-acetyl group (MOCAc) in the substrate, MOCAc-Ala-Pro-Lys-PhePhe-Arg-Leu-Lys(Dnp)-NH2 (Peptide Institute Inc., Japan). The principle of the assay is that the MOCAc group on this substrate is efficiently quenched by the 2,4-dinitrophenyl (DNP) group before proteolysis (Figure 2.5). When proteinase A (Pr A) cleaves the Phe-Phe bond, the fluorescence at excitation 328 nm and emission 393 nm increases several hundred fold and the proteinase A activity can be estimated from the intensity of this fluorescence. The wort or beer sample was centrifuged at 2, 800 g for 10 min and the supernatant used for the Pr A assay. The Pr A preparation (40 µL) was pipetted into an Eppendorf tube and 500 µL McIlvaine buffer (0.2 M disodium hydrogen phosphate plus 0.1 M citric acid, 67 Chapter 2 Materials and Methods pH 4.5), 456 µL dH2O and 4 µL MOCAc substrate (1 mM substrate in DMSO) were added. The mixture was gently mixed and incubated at 30C for 30 min. The reaction was terminated by heating at 80C for 5 min, cooled on ice for 15 min and then centrifuged at 6,000 g for 1 min. The reaction mixture was diluted 10 fold using dH2O and the fluorescence was measured in a quartz cuvette with a 10 mm light path in a Perkin-Elmer fluorescence spectrometer model 203 (Perkin-Elmer, USA). Yeast proteinase A was used as a standard (Sigma, catalogue number P8892). a) CH2CO-Ala-Pro-Ala-Lys-Phe-Phe-Arg-Leu-Lys(DNP)-NH2 MeO o o Fluorescence is quenched by DNP Proteolysis by Proteinase A b) CH2CO-Ala-Pro-Ala-Lys-Phe- -Phe-Arg-Leu-Lys(DNP)-NH2 o MeO o No Function High Fluorescence Figure 2.5. Principle of the extra cellular proteinase A assay method using MOCAc-Ala-Pro-Lys-PhePhe-Arg-Leu-Lys(DNP)-NH2 (Kondo et al., 1998). 2.23 Fluorescence microscopy and confocal imaging Open and closed perfusion was performed with a POC (Perfusion, Open and Closed cultivation) chamber-system assembled on a heating insert P that was controlled by a Tempcontrol 37 (Helmut Saur Laborbedarf, Germany) temperature controller. The flow of media was maintained using an Amersham Peristaltic Pump P-1. Live cells were immobilised by the use of a 0.22 µm pore size isopore polycarbonate filter membrane 68 Chapter 2 Materials and Methods (Millipore, Watford, United Kingdom) or a solution of 1 mg/mL concanavalin A in dH2O spread on the microscope glass slide. Confocal fluorescence microscopy was conducted using an inverted microscope (DM IRE2, Leica Microsystems, Germany) with a Laser Scanning Confocal Microscope (LSCM) attachment (TCS SP2 AOBS, Leica Microsystems). The system acquired images with a 63x objective (1.4 NA). The pictures were processed by Leica Confocal Software TCS SP2 Version 2.5.1227a. Data coming from the photomultiplier tubes (PMTs) was digitised with 8-bit precision for display. Multiple PMTs and fluorophores allowed the overlay of pseudo-coloured images to produce one composite multicoloured image. Fixation of yeast cell samples for confocal imaging and flow cytometry Samples of approximately 2 mL of cell suspension were retrieved and immediately immersed in 10 mL cold 70% (v/v) reagent grade ethanol and incubated for at least 4 h at 4°C. Cells treated in this manner can be stored at 4°C for up to one month prior to analysis. The fixation in ethanol enables the permeation of large dye molecules through the cell membrane. The membrane of living cells is able to selectively permit passage or exclude certain substances. Dead cells lack this semi-permeability and easily allow substances such as large fluorescence molecules to pass into the cell. The surface structure of the cells is deformed by means of water removal. The denaturation does not alter the composition of protein compounds and many intracellular macromolecules such as DNA and glycogen are preserved during the alcoholic fixation (Gharton et al., 1975). Sampling of viable yeast cells for staining procedures for confocal imaging and flow cytometry An appropriate volume (approximately 10 mL) of yeast cell suspension was retrieved and staining was conducted immediately. 2.24 Staining of physiological parameters in yeast cells for confocal imaging and flow cytometry 2.24.1 Staining of glycogen with acriflavine 69 Chapter 2 Materials and Methods The specific staining of glycogen with acriflavine was carried out according to the procedures of Gharton et al. (1975); Meyer et al. (1977) and Hutter et al. (2000) with minor modifications. Preparation of Schiff reagent: Concentrated HCl (10 mL) was diluted to 100 mL with dH2O. Acriflavine (0.5 g) was dissolved in 15 mL diluted HCl. K2S2O5 (0.5 g) was dissolved in 85 mL dH2O. The K2S2O5 solution was added to the acriflavine solution. After 24 h 300 mg charcoal was added. The solution was mixed for 2 min, filtered through a fluted filter and stored at 4°C and protected from light. Staining procedure: PBS buffer (4 mL) was added to 2 mL yeast suspension in 70% (v/v) ethanol. The yeast suspension was centrifuged for 10 min at 6,000 rpm. The supernatant was discarded and the washing step repeated. The resulting cell pellet was re-suspended in 1 mL periodic acid solution (0.5 g H5IO6 in 100 mL dH20), mixed and incubated at room temperature for 10 min. The suspension was washed with 4 mL PBS buffer. One mL diluted Schiff reagent (1 mL PBS + 10 µL Schiff reagent) was added. The suspension was incubated for 1 h at room temperature in the dark and washed twice with 4 mL PBS buffer. The cell pellet was re-suspended in 2 mL PBS buffer. The dye was excited with the 488 laser line of an argon-ion laser and the fluorescent emission was collected at a wavelength of λ = 498–560 nm. 2.24.2 Staining of neutral lipids with the fluorescent dye nile red The neutral lipids in yeast cells were stained with nile red according to the method of Hutter et al. (1996). The highly lipophilic benzophenoxazone dye nile red intercalates in lipid droplets and stains neutral lipids inside the cell and with a lower intensity also the phospholipids of the cell membrane. Figure 2.6 depicts the molecular structure of the nile red dye and Figure 2.7 shows the Stoke’s shift in wavelength between excitation and emission for nile red bound to a phospholipid bi-layer membrane. PBS buffer (4 mL) was added to 2 mL yeast suspension in 70% (v/v) ethanol. The yeast suspension was centrifuged for 10 min. at 6,000 rpm. The supernatant was discarded and the washing step repeated. The resulting cell pellet was re-suspended in 2 mL PBS and 40 µL nile red solution (1 mg/mL nile red in acetone) was added. 70 Chapter 2 Figure 2.6. Structure of nile red. Materials and Methods Figure 2.7. Absorption and fluorescence emission spectra of nile red bound to phospholipid bi-layer membranes (Hutter et al., 1996). The suspension was vortexed and incubated for 30 min at room temperature in the dark. The fluorescence emission reached its maximum after 30 min of incubation and decreased after 45 min. The equilibrium between free nile red dye molecules in buffer solution and the dye bound intracellularly was impaired by the hydrophobic nature of the dye. Therefore, the exact staining time must be observed. The dye was excited with the 543 laser line of a green He/Ne laser and the fluorescence emission was collected at a wavelength of λ = 548-660 nm. Neutral lipids inside the cytoplasm of yeast cells emit goldyellow light. 2.24.3 Staining of trehalose with the fluorescent dye concanavalin A-fluorescein The lectin-fluorochrome-conjugate concanavalin A-fluorescein was used to stain the yeast cells for trehalose. Concanavalin A belongs to the class of lectins (from Canavalia ensiformis). Concanavalin A selectively binds to α-mannopyranosyl and α-glucopyranosyl residues on the surface of yeast cells. The staining was conducted according to the method of Hutter et al. (2003). PBS buffer (4mL) was added to 2 mL yeast suspension in 70% (v/v) ethanol. The yeast suspension was centrifuged for 10 min at 6,000 rpm. The supernatant was discarded and the washing step repeated. The resulting cell pellet was re-suspended in 2 mL PBS buffer and 20 µL concanavalin A-fluorescein solution was added. The suspension was incubated for 20 min at room temperature in the dark. The dye was excited with the 488 laser line of an argon-ion laser and the fluorescent emission was collected at a wavelength of λ = 500-590 nm. 71 Chapter 2 Materials and Methods 2.24.4 Staining of bud scars with WGA-fluorescein Bud scars were visualised on the surface of yeast cells by staining with the fluorescent dye WGA (Wheat Germ Agglutinin)-fluorescein (Hutter and Nitzsche, 2002). Chitin-rich regions on the surface of yeast are stained and highly fluorescent yeast cells can be distinguished from weakly fluorescent populations. High fluorescent signals imply the existence of many old or dead cells in the cell population. A relatively low fluorescence indicates a young and dynamic cell population. PBS buffer (4 mL) was added to 2 mL yeast suspension in 70% (v/v) ethanol. The yeast suspension was centrifuged for 10 min at 6,000 rpm. The supernatant was discarded and the washing step repeated. The resulting cell pellet was resuspended in 1 mL PBS buffer. WGA-fluorescein solution (30 µL) (1 mg/mL WGA-fluorescein in PBS buffer) was added. The suspension was vortexed and incubated for 1 h at room temperature in the dark. The suspension was centrifuged, the supernatant discarded and the cell pellet resuspended in 2 mL PBS buffer. The dye was excited with the 488 laser line of an argon-ion laser and the fluorescent emission was collected at a wavelength of λ = 500-580 nm. 2.24.5 Fluorescent staining of yeast DNA with propidium iodide The DNA content was visualised with propidium iodide (Figure 2.8) following RNAse digestion according to the procedure of Hutter (1978) and Hutter and Eipel (1978) with minor modifications (Müller, 1992). Figure 2.8. Structure of propidium iodide. Figure 2.9. Absorption and fluorescence emission spectra of propidium iodide bound to DNA (Hutter and Eipel, 1978). 72 Chapter 2 Materials and Methods PBS buffer (4mL) was added to 2 mL yeast suspension in 70% (v/v) ethanol. The yeast suspension was centrifuged for 10 min at 6,000 rpm. The supernatant was discarded and the washing step repeated. The resulting cell pellet was re-suspended in 1 mL RNAse solution (1 mg/mL RNAse A in PBS buffer pre-warmed to 37°C) and incubated for 1 h at 37°C. The enzyme reaction was terminated by addition of 5 mL cold PBS buffer. The suspension was centrifuged and the supernatant discarded. The cell pellet was re-suspended in 2 mL PBS buffer and 200 µL PI solution was added. The suspension was incubated for at least 1 h at 37°C in the dark (incubation overnight is preferable). The dye was excited with the 488 laser line of an argon-ion laser and the fluorescent emission was collected at a wavelength of λ = 550-710 nm. Propidium iodide (Figure 2.8) belongs to the chemical class of the phenantridium derivatives and is the most common red fluorescent nuclear stain. It is a large planar molecule that is membrane impermeant, and thus does not stain living cells. Propidium iodide is commonly used for identifying dead cells in a population and as a counter stain in multicolour fluorescent techniques. Phenantridium derivatives are dyes that bind to doublestranded DNA by intercalating between super-imposed bases with little or no sequence preference and with a stoichiometry of one dye per 4–5 base pairs of DNA. It also binds to the double-stranded t-RNA inside the cytoplasm, necessitating removal of RNA by RNAse digestion in order to distinguish between RNA and DNA. Once the dye is bound to nucleic acids, its fluorescence is enhanced 20- to 30-fold. The fluorescence light efficiency of propidium iodide is higher than that of any other phenantridium derivatives. It was first used for the DNA analysis of mammalian cells by Crissman and Tobey (1974). Hutter and Eipel (1978) first used this fluorochrome for the fluorescent analysis of yeast cells. When bound to nucleic acids, the absorption maximum for propidium iodide is 535 nm, and the fluorescence emission maximum is 617 nm (Figure 2.9). The stained nucleus emits bright red light. Although its molar absorptivity (extinction coefficient) is relatively low, propidium iodide exhibits a sufficiently large Stokes shift to allow simultaneous detection of nuclear DNA and fluorescein-labelled molecules. 2.24.6 Intracellular proteinase staining with BODIPY-FL-casein The staining for intracellular proteinase was conducted according to the method of Hutter et al. (2005). The EnzChek® Proteinase Assay Kit E-6638, supplied by Molecular Probes contains the lyophilised BODIPY FL substrate and the 20x digestion buffer. 73 Chapter 2 Materials and Methods The staining procedure was conducted with a casein substrate labelled with quenched BODIPY® dye, which is a substituted 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative (Haugland and Kang, 1988; Jones et al., 1997). This casein-dye conjugate provides spectrally distinct substrates for detecting metallo-, serine, acid and sulphydryl proteinases. The assay kit contains casein derivatives that are heavily labelled with the pH-insensitive greenfluorescent BODIPY® FL dye. These conjugates are highly quenched and typically exhibit only <3% of the fluorescence of the corresponding free dyes. Proteinase catalysed hydrolysis releases highly fluorescent BODIPY FL-dye labelled peptides and the accompanying increase in fluorescence is proportional to proteinase activity (see Figure 2.10). The detection of intracellular proteinase activity gives an early indication of apoptotic events and allows improved assessment of the physiological state of a yeast population (Dernby, 1917). BODIPY FL casein and their hydrolysis products show green fluorescence with maximal excitation at about 503 nm and emission maxima near 512 nm (Jones et al., 1997). Proteinases Figure 2.10. Principle of detection of intracellular proteinases using the BODIPY FL fluorescent kit (Haugland and Kang, 1988). Preparation of BODIPY FL casein stock solution: A 1.0 mg/mL stock solution of the BODIPY FL casein was prepared by adding 0.2 mL PBS buffer directly to one of the vials containing 200 µg of the lyophilised substrate. Sufficient time at room temperature was allowed for the substrate to dissolve and it was stored at 4°C in a dark bottle. Preparation of working-strength digestion buffer: Concentrated 20 x digestion buffer (2.5 mL) was diluted with dH2O to a final volume of 50 mL and stored at 4°C. Preparation of BODIPY FL casein working solution: A 10 µg/mL working solution of the BODIPY FL casein was prepared by adding 0.2 mL of the BODIPY FL casein stock solution to 19.8 mL of the working-strength digestion buffer and stored at 4°C in a dark bottle. 74 Chapter 2 Materials and Methods Staining procedure: PBS buffer (4 mL) was added to 2 mL yeast suspension in 70% (v/v) ethanol. The yeast suspension was centrifuged for 10 min at 6,000 rpm. The supernatant was discarded and the washing step repeated. The resulting cell pellet was re-suspended in 1 mL working-strength digestion buffer and 1 mL of the BODIPY FL casein working solution was added. The samples were incubated for 1 h at room temperature in the dark. The dye was excited with the 488 laser line of an argon-ion laser and the fluorescent emission was collected at a wavelength of λ = 498–550 nm. 2.24.7 Staining of the vacuolar membrane with FM® 4-64 The lipophilic styryl dye FM 4-64 (N-(3-triethylammoniumpropyl)-4-(6-(4(diethylamino) phenyl) hexatrienyl) pyridinium dibromide) (Figure 2.11) has been reported to selectively stain yeast vacuolar membranes with red fluorescence (Vida and Emr, 1995). This lipophilic dye is an important tool for visualising vacuolar organelle morphology and dynamics. It is a vital stain, which means it exhibits fluorescence only in living cells, so cells cannot be fixed then stained, nor can they be stained then fixed. FM 4-64 does not permeate cell membranes. Rather, FM 4-64 intercalates into the plasma membrane of yeast cells and is then taken into the cells by an endocytic mechanism (Vida and Emr, 1995). This dye selectively labels the membrane of the intracellular organelles along the endocytic pathway since it is fluorescent only when inserted into membranes. During a time-course of FM 4-64 staining, the dye initially stains the yeast plasma membrane, then the cytoplasmic intermediate endosomal compartments and finally the vacuolar membrane (Vida and Emr, 1995). Figure 2.11. Structure of the fluorescent vacuolar membrane stain FM-4-64 (Vida and Emr, 1995). The internalisation of the lipophilic styryl dye FM 4-64 by endocytosis was used to stain the vacuolar membrane. The staining procedure is a modification of the methods of Vida and Emr (1995) and Meaden et al. (1999). Preparation of FM 4-64 stock solution: The dye (1 mg) was dissolved in 100 µL DMSO (16mM). The solution was stored in the freezer at -20°C in an Eppendorf tube. 75 Chapter 2 Materials and Methods Preparation of FM 4-64 working solution: To 2.5 µL stock solution 50 µL of DMSO was added (800 µM) and stored at -20°C in an Eppendorf tube. Staining procedure: A 50 µL aliquot of viable log-phase yeast cells (0.5-1.0 Optical Density Units) was transferred to an Eppendorf tube and centrifuged at 12,000 rpm for 5 min. The supernatant was aspirated. The resulting cell pellet was re-suspended in 0.5 mL YPD medium and 2.5 µL FM 4-64 working solution giving a final dye concentration of 4 µM. The cells were incubated on a shaker at 30°C for 20 min at 150 rpm. One mL YPD medium was added. The suspension was centrifuged at 12,000 rpm for 5 min. The supernatant was aspirated. The cell pellet was re-suspended in 1 mL YPD. The suspension was transferred to a centrifuge tube and 4 mL YPD medium was added. The suspension was shaken at 30°C for 60 min. The centrifuge tube was spun for 15 min at 6,000 rpm. The supernatant was aspirated and the cell pellet re-suspended in 1 mL sterile water. The suspension was transferred to an Eppendorf tube and centrifuged at 12,000 rpm for 5 min. The supernatant was aspirated and the cell pellet re-suspended in 50 µL YNB medium. A glass slide was coated with 10 µL of a 1:1 mixture of 1 mg/mL concanavalin A in dH2O. The mixture was spread with the side of a pipette tip evenly over the slide and allowed to air dry. A 7 µL aliquot of the yeast suspension was dotted onto the concanavalin A-coated glass slide and covered with an 18 mm x 18 mm number 1 cover slip ensuring that all air bubbles were removed. The dye was excited with the 543 laser line of a green He/Ne laser and the fluorescent emission was collected at a wavelength of λ = 637–750 nm. 2.25 Measurement of Reactive Oxygen Species (ROS) using Electron Spin Resonance (ESR) ESR experiments were carried out in a Bruker Biospin ESR instrument. A 10 mL aliquot of beer was degassed by passage through a Whatman no. 1 filter paper. The beer was equilibrated to room temperature; 10 mL was transferred to a brown vial, and 0.5 mL of PBN solution (1.77 g/10 mL 98% ethanol) was added. The vials were covered with aluminium foil, and placed in a heating block at 60oC. Over the ensuing period, the ESR signal arising from the PBN-OH radical was measured. See Figure 2.12 for a description of experimental set up. Up to 10 samples were measured during any one analysis run. Beer samples were used on the day of opening the container. In some cases the beer was treated with sodium metabisulphite and/or EDTA in various concentrations, which were made up fresh, each day in distilled water and stored at refrigerator temperature. Lag time and T150 data points were acquired using the Bruker Biospin's software. However, the lag times were also calculated after plotting the data using an excel spreadsheet program. In 76 Chapter 2 Materials and Methods some cases it was found that the instrument's software was unreliable when any one of the data sets failed to behave in a predicted fashion. That is, showing a typical hyperbolic signal accumulation over time. The Principles of Electron Spin Resonance Continued... Beer sample PBN (spin-trapping reagent) PBN PBN OHPBN OH- OHPBN 60°C OH- PBN OH- Figure 2.12. Principle of ESR measurement for the detection of Reactive Oxygen Species (ROS). 2.26 The Peroxide Challenge Test (PCT): A novel assay for predicting beer flavour stability A 25 mM luminol stock solution in DMSO was prepared and then stored at –20oC. The luminol reagent was prepared by adding thawed luminol stock solution to a final concentration of 0.1 mM and peroxidase to a final concentration of 1.0 U/mL in Kolthoff buffer, pH 8.5 (50 mM Na3B4O7, 100 mM KH2PO4). A 180 µL aliquot of beer or process sample was added to 20 µL of peroxide (4.0, 2.0, 1.0, 0.8, 0.6, 0.4 and 0.2 mM) and incubated for 30 min at room temperature (Figure 2.14). A 20 µL aliquot of the beerperoxide mixture was transferred to a 96 well plate (Wallac isoplates, Perkin Elmer) and 180 µL luminol reagent was injected (Figure 2.15). Luminescence intensity detected with a Wallac Victor2 1420 Mulitlabel Counter, Perkin Elmer with an automated injection facility. Figures 2.13, 2.14 and 2.15 show the experimental procedure in detail. Chemiluminescence, as counts per sec (CPS), was measured at room temperature. Kinetic readings of each well 77 Chapter 2 Materials and Methods were taken in the first 15 sec following injection of the luminescing reagent. Specifically, 10 readings were recorded for each well, with a 0.5 sec interval between each reading. The 10 Peroxide Challenge Test (PCT) – Experimental readings were averaged and the H2O2 generation activity in CPS plotted for each sample. 180 µL beer 20µL Beer-PO 20µL PO 180µL light up solution Luminescence 30 min RT Figure 2.13. Principle of peroxide challenge test for measuring beer flavour stability. Peroxide 400 μM 200 μM 100 μM 80 μM 60 μM 40 μM 20 μM 180μL Beer +20μL H2O 200μL H2O 0 μM 180μL H2O +20μL PO Beer W Beer X Beer Y Beer Z 30 minutes at RT then transfer 20μL to a new plate.. Figure 2.14. Step one of the PCT analysis. Beer sample (180 μL) was added to 20 μL of hydrogen peroxide solution (0-400 μM) and incubated for 30 min at room temperature. Appropriate blanks were included. 78 Chapter 2 Materials and Methods Peroxide 400 μM 200 μM 100 μM +180μL Luminol Reagent 80 μM 60 μM 40 μM 20 μM +20μL Beer/H2O 20μL H2O 0 μM +20μL PO/H2O Beer W Beer X Beer Y Beer Z Figure 2.15. Step two of the PCT analysis. Beer/peroxide mixture (20 μL) was transferred to a fresh 96 well plate. Luminol reagent (180 μL) was added to each well individually and luminescence detected. In the yellow wells the beer antioxidants were able to quench all the added peroxide and thus no lumiscence signal was detected. The red wells indicate the lowest concentration of peroxide added that lead to a detection of luminescence. This is called the peroxide breakthrough point. The peroxide breakthrough point for the imaginary beers W, X, Y and Z (wells marked in red) is a direct measure for the antioxidant potential of these beers. From this point onwards increasing peroxide concentrations added lead to an increase in the luminescence detected (wells in blue). 2.27 3,3’,5,5’-tetramethylbenzidine (TMB) detection for rapid analysis of beer protein thiol concentration TMB is the most sensitive chromogenic peroxidase substrate for Western and Dot Blotting applications. Detection limits are significantly increased as compared to other chromogenic membrane substrates. TMB produces a dark blue precipitate upon reaction with avidin Horseradish Peroxidase (HRP). 79 Chapter 2 Materials and Methods TMB (1mg) was added to 10 mL phosphate citrate solution. Then 2 µL of hydrogen peroxide (30%) was added. This reagent was made up fresh on the day of use and kept refrigerated and protected from light. The TMB was purchased in 1mg tablet form. 2.28 Labelling of protein thiols using MPB (N'-(3-maleimidylpropionyl) biocytin) MPB labelling: Beer samples were mixed 4:1 with 0.2 M sodium phosphate buffer pH 8.0. MPB (N'-(3-maleimidylpropionyl) biocytin) was added to a final concentration of 0.5 mM and allowed to react for 30 min at room temperature before un-reacted reagent was quenched by the addition of 2 mM DTT. Samples were then analysed by SDS PAGE/Western Blot, developed with avidin-peroxidase and reactive proteins were visualised by chemiluminesence detection. 2.29 Total thiol determination in beer using DTNB 2 mM cysteine standard: Cysteine (0.012 g) was dissolved in 50 mL sterile ddH2O. This standard solution was stored in the fridge and was used over a few days. DTNB reagent (55'-dithiobis-(2-nitrobenzoic acid)): Sodium phosphate (0.05 M), pH 8.0, 1 mM EDTA, 1.5% SDS, 1 mM DTNB. DTNB reagent is unstable and was prepared fresh as required. A 1 mM cysteine working solution from the 2 mM cysteine standard was prepared and serially diluted to 15.6 µM in ddH2O in Eppendorf tubes. A 20 µl aliquot of each dilution of the standard was added in triplicate to a 96-well plate, 20 µL of ddH2O was added as both zero and blank. The unknowns were added to the plate in triplicate. For beer samples, 20 µL of undiluted beer was used. After the addition of the standards and the unknowns to the plate, 180 µL of DTNB reagent was added. The plate was incubated at room temperature in the dark for 20 min. The plate was mixed and read at 412 nm against the blank. 2.30 Thiol determination in beer proteins using an enzyme-linked immunosorbent assay (ELISA) The following outline describes a typical protocol developed for the rapid detection of protein thiol groups employing an ELISA using MPB as the thiol specific reagent. 80 Chapter 2 Materials and Methods 2.30.1 Preparation of reduced/oxidised Bovine Serum Albumin (BSA) as standard protein Reduced BSA 1. A 20 mg/L BSA solution in PBS-EDTA buffer (2 mM EDTA) was prepared and 1,4-Dithio-DL-threitol (DTT) was added to a final concentration of 20 mM. 2. Samples were incubated for 15 min at 37ºC. 3. The spin columns were regenerated with 500 µL PBS-EDTA for 2 min at 1,000 g. This was repeated 5 times. 4. BSA solution (100 µL) was loaded onto the spin column and spun for 4 min at 1,000 g. 5. The filtrate containing the reduced BSA was collected and stored at 4˚C. 6. The reduced BSA solution was suitably diluted and the protein content estimated by measuring the absorption of the BSA solution at λ=280 (A280). Oxidised BSA 1. A 20 mg/L BSA solution in PBS-EDTA (2 mM EDTA) was prepared 2. The BSA solution was suitably diluted and the protein content estimated by measuring the absorption of the BSA solution at λ=280 (A280). It was approximated that the BSA used was fully oxidised. 2.30.2 Oxidised and reduced BSA as standard protein: Estimation of protein thiol content using DTNB This measurement was carried out as described in 2.29 with some minor modifications: Oxidised BSA (40 µL) was placed in a 96 well plate; 160 µL of the DTNB reagent was added and the measurement conducted as described in 2.29. The oxidised BSA should contain around 0.58 µM thiol groups. The reduced BSA preparation was diluted 1:3. Reduced BSA (40µL) was placed in a 96 well plate and 160 µL of the DTNB reagent added. The analysis was conducted as described in 2.29. The reduced BSA contained approximately 13 µM of thiol groups. 81 Chapter 2 Materials and Methods Stability and storage of the BSA standards In order to keep the reduced BSA preparation in the reduced state for a prolonged period, the pH of the BSA preparation was adjusted to pH 6.5, from the original pH 7.2 to slow down oxidation. Furthermore, the reduced BSA standard was stored at 4ºC. However, despite these precautions the thiol groups in the reduced BSA were slowly oxidised. Thus it is recommended that before using the standards, the DTNB assay should be conducted to establish the concentration of thiol groups. After two weeks, the reduced BSA was fully oxidised and no thiol groups could be detected and thus a fresh reduced BSA standard was prepared. Also a fresh oxidised BSA standard was prepared on this occasion since microbial growth in the oxidised BSA standard could produce un-usable results in the ELISA determination of thiol groups. Preparation of mixtures of reduced BSA and oxidised BSA The reduced and oxidised BSA preparations were diluted to a concentration of 10 µg/mL. Mixtures of reduced and oxidised BSA were prepared to produce solutions of BSA containing 100%, 80%, 60%, 40%, 20% and 0% reduced BSA/oxidised BSA: A, B, C, D, E, F (see Table 2.5). Table 2.5. Mixtures of reduced and oxidised BSA to create a standard curve for measuring thiol content in beer proteins. A B C D E F BSA (Reduced) 100 80 60 40 20 0 BSA (Oxidised) 0 20 40 60 80 100 Preparing the MultiScreenHTS 96 well plates The MultiScreen plates were placed on the vacuum manifold. 100% methanol (HPLC grade) (100 µL) was added to each well to pre-wet the polyvinylidene fluoride (PVDF) membrane. The methanol was removed by pulling it through the membrane using the vacuum (Pull It through: “PIT”). 20% methanol in sodium phosphate buffer (0.02 M, pH 8) (100 µL) was added and pulled through the membrane. Each well was washed twice with 200 µl Sodium Phosphate buffer (0.02 M, pH 8) and PIT. Sodium phosphate buffer 82 Chapter 2 Materials and Methods (0.02 M, pH 8) (100 µL) was added to row A, columns 1-12 (non-coated wells are the plate blanks). BSA standards (100 µL) were added to the appropriate wells. The plate was incubated for 20 min at room temperature and PIT. The plate was washed 4 times with 200 µL Tris buffered saline (TBS) and PIT. One hundred µL of 50 µm MPB in sodium phosphate buffer (0.02 M, pH 8) was added and incubated for 15 min at room temperature, PIT. The plate was washed twice with sodium phosphate buffer (0.02 M, pH 8) and PIT. One hundred µL of 0.25mM DTT (dithiothreitol) in sodium phosphate buffer (0.02 M, pH 8) was added and incubated for 5 min at room temperature, PIT. The plate was washed 4 times with TBS-Tween, PIT. Blotto (5%) (5% dried milk (w/v) in TBS) (200 µL) was added to the plate and incubated for 15 min at room temperature. After the incubation period the plate was removed from the vacuum manifold and the Blotto was poured out. Avidin horseradish peroxidase diluted 1/3,000 with the 5% Blotto was added (100 µL) to each well and incubated for 60 min at room temperature. The solution was removed and the plate washed 3 times with TBS-Tween, PIT. Another standard opaque 96 well plate was placed underneath the MultiScreen plate in the vacuum manifold so that the filtrate from the Multiscreen plate flowed into the corresponding well of the 96 well plate positioned underneath (collection plate). TMB light up solution (125 µL) was added to all wells of the Multiscreen plate and incubated at room temperature (see also 2.27). The colour reaction was stopped by adding 125 µL of 1 M H2SO4. The blue liquid was pulled through into the collection plate. The absorption of the collection plate was read at λ=450 nm. 2.31 Detection of protein carbonyl groups with an enzyme linked immunosorbent assay (ELISA) 2.31.1 Preparation of oxidised and reduced BSA standards Bovine serum albumin (BSA) was dissolved in 20 mM Tris, pH 7.4, at a concentration of 1 µg/µL, for use as standards and for testing the specificity of the method. The proteins were analysed in three different oxidation states: (A) native proteins were assayed directly, without any oxidation or NaBH4 reduction; (B) proteins were oxidised by treatment with H2O2 and Fe2+; and (C) proteins were reduced by treatment with NaBH4 prior to assay. Oxidised proteins were prepared by incubation with H2O2 and ferrous sulphate (1 mM each) for 10 min at room temperature. The oxidised protein was then divided into two equal aliquots and both were precipitated with an equal volume of 20% trichloroacetic acid (TCA). After centrifugation at 1,000 g for 10 min, the 83 Chapter 2 Materials and Methods supernatants were decanted and the protein pellets were washed three times with TCA to remove any excess H2O2 and ferrous sulphate. One of the protein pellets was re-suspended in 85.7 mM Tris, 0.857 mM EDTA, 20 mM NaOH buffer, pH 8.5. The second protein pellet was reduced by re-suspension in 85.7 mM Tris, 0.857 mM EDTA, 20 mM NaOH buffer, pH 8.5, and containing 20 mM NaBH4 and incubated at 37°C for 30 min. The native proteins were neither oxidised nor reduced, but the samples were precipitated, washed with TCA, and re-suspended in Tris-EDTA buffer without NaBH4. The protein concentrations of the re-suspended preparations were determined using a commercially available protein assay based on the method of Bradford. All samples were stored frozen at -20°C prior to analysis. A series of BSA standards of the same protein concentration but with different carbonyl contents were prepared by mixing oxidised BSA and sodium borohydride-treated BSA in different proportions (see Table 2.5). These standards were analysed with the colorimetric technique to determine their carbonyl content and were included as standards in all subsequent experiments. Furthermore, some experiments were carried out with a blank containing no carbonyl groups. 2.31.2 Blanking carbonyl reactivity with sodium borohydride For a blanked determination, one aliquot of the BSA was prepared in the usual manner (85.7 mM Tris, 0.857 mM EDTA, 20 mM NaOH buffer, pH 8.5), and a second BSA aliquot were prepared in the same Tris–EDTA buffer containing 20 mM NaBH4. Since NaBH4 reduces carbonyl groups to alcohols, such a treatment was expected to eliminate any immunostaining due to carbonyl groups. Thus, the difference between the staining intensities of a NaBH4-treated and an untreated aliquot should constitute a specific measure of the carbonyl content of the BSA. 2.31.3 Determination of protein oxidation with the standard colorimetric technique for detection of carbonyl groups The carbonyl content of oxidised BSA standards was determined in triplicate using a well-established colorimetric assay (Levine et al., 1990; Levine et al., 1994). The proteins contained in 500 mL beer were precipitated with an equal volume of 20% trichloroacetic acid. The supernatant was discarded. To the pellet 500 µL of 0.2% DNPH (in 2 N HCl) was added and incubated for 1 h in the dark. Corresponding blanks were prepared using 2 N HCl alone. The derivatised proteins and their blanks were precipitated by the addition of 84 Chapter 2 Materials and Methods an equal volume of 20% TCA and centrifugation at 11,000 g for 3 min. The supernatants were discarded and the pellets were washed three times with 1 mL of ethanol:ethyl acetate (1:1 v/v). The samples were vortex mixed periodically during each 10-min organic solvent wash and then centrifuged (11,000 g for 3 min). The final pellets were re-suspended in 0.6 mL of 6 M guanidine–HCl, and the carbonyl contents were determined by measuring the absorbance of the DNP-hydrazones at 370 nm. Results were calculated as nmol carbonyl/mg protein using an extinction coefficient of 22,000 M-1 cm-1. 2.31.4 DNPH derivatisation after absorbing the beer proteins on the PVDF membrane: “On the plate derivatisation” Sample preparation The BSA standards were diluted with 85.7 mM Tris, 0.857 mM EDTA, 20 mM NaOH, pH 8.5 buffer to a protein concentration of approximately 50 µg/mL. The samples and standards were kept on ice until application to the 96-well MultiScreenHTS Plates (equipped with a hydrophobic Immobilon-P PVDF membrane, 0.45 µm at the bottom of the wells). Preparing the PVDF membrane and blotting The PVDF membrane at the bottom of the 96 well Multiscreen plate was prepared by wetting it with 200 µL 100% MeOH and then adding 200 µL of 20% MeOH–80% Trisbuffered saline solution (TBS, 20 mM Tris-base, 500 mM NaCl, pH 7.4) for 5 min. Diluted BSA solution (5 µg protein) (100 µL) was applied to each well. One hundred µL of the beer samples was applied undiluted to the wells. The samples were left undisturbed on the membrane for 20 min and then a vacuum was applied to the vacuum manifold until all of the liquid above the membrane had just disappeared. DNPH derivatisation of the membrane MeOH 100% (200 µL) was applied to each well and left undisturbed for 10 min. Sequentially, the membrane was washed with 200 µL 20% MeOH–80% TBS and then 200 µL 2 N HCl followed by 200 µL 2,4-dinitrophenylhydrazine (100 µg/mL) in 2 N HCl for exactly 5 min. The duration of this derivatisation step is critical. The membrane was washed three times with 200 µL 2 N HCl, and seven times with 200 µL 100% MeOH. Immunostaining 85 Chapter 2 Materials and Methods The membrane was washed two times with 200 µL TBS and non-specific protein binding sites were blocked with 5% dried milk (w/v) in TBS (Blotto) for 1 h. The membrane was washed three times in 200 µL TBS containing 0.05% (v/v) Tween 20. The membrane was incubated for 18 h at 4°C with the primary antibody (anti-DNP antibody) solution consisting of a 1:25,000 dilution of the anti-2,4-dinitrophenol antibody in TBS containing 5% dried milk and 1% Tween 20 (antibody diluent). The membrane was washed five times with 200 µL of the same TBS/milk/Tween solution used as the antibody diluent. The membrane was then incubated with a 1:5000 dilution of the secondary antibody (avidin-horseradish peroxidase) in antibody diluent for 1 h at room temperature. The membrane was washed six times with 200 µL TBS/milk/Tween solution used as the antibody diluent and once with TBS containing 0.05% Tween. TMB light up solution (125 µL) was added to all wells of the Multiscreen plate and incubated at room temperature until a sufficient amount of blue colour developed. The colour reaction was stopped by adding 125 µL 1 M H2SO4 to all wells. The blue liquid was pulled through into the collection plate and the collection plate was read at λ=450 nm. 2.32 Protein extraction and purification from beer wort, hot water extracts and other liquid process samples Ammonium sulphate (206 g) was added to wort, beer, raw material hot water extracts or other liquid process samples at 4˚C (400 mL). This was allowed to dissolve and was mixed for a further 30 min. The liquid was transferred to 250 mL centrifuge bottles which were centrifuged at 7840 rpm for 10 min (10,000 g) in a Sorval RC24 centrifuge (Du Pont). The supernatant was decanted. The pellet was resuspended in distilled water using a glass homogeniser. The liquid was then dialysed in Visking tubing with a Molecular Weight Cut Off of 12 – 14 kDa (Medicell Industries Ltd.). The tubing was boiled in water and washed before use to remove the preservative coating. The sample was retained in the tubing by knotting both ends of the dialysis tube. Samples were dialysed against distilled water at 4˚C for two days with the water being changed twice. Samples were then removed from the tubing and placed on metal trays for freeze drying. The trays were placed in a freezer until frozen and then moved into the freeze dryer (Super Modulyo Freeze Dryer Edwards) and dried for three days. The proteinaceous material was recovered from the trays, weighed and stored at 4˚C). 2.33 Gel electrophoresis and visualisation of protein bands using silver staining 86 Chapter 2 Materials and Methods Gel electrophoresis techniques were used to visualise polypeptides in wort, beer or purified protein samples. The protein bands were visualised by silver staining or by staining the gel with Coomassie blue dye. Sample preparation for SDS polyacrylamide gel electrophoresis (SDS PAGE) of purified protein samples Approximately 1 mg of sample was placed in a screw-cap Eppendorf tube containing 400 µL water and mixed. To this 400 µL sample buffer was added. Sample Buffer Water 24 mL Tris HCl, 0.5 M, pH 6.8 6 mL (6.055g Tris in 100mL water, buffer to pH 6.8, make up to 100mL) Glycerol 5 mL Sodium Dodecyl Sulphate (SDS) (10% w/v) 10 mL Bromophenol blue (0.5% w/v) 2.5 mL To 6,650 µL sample buffer, 350 µL mercaptoethanol was added immediately before use. The tubes were sealed and boiled in a waterbath for 10 min. If the samples were not to be used immediately, they were stored in a freezer at -20ºC, but boiled again for 5 min before loading. Molecular weight standards (BioRad broad range standards) were run on each gel. Standard (5 µL) was mixed with 95 µL sample buffer and boiled for 10 min. Sample preparation for SDS polyacrylamide gel electrophoresis (SDS PAGE) of beer and wort samples Sample (400 µL) was placed in a screw-cap Eppendorf tube and 400 µL of the sample buffer was added and mixed. The mixture was boiled for 10 min in a waterbath before application to the gel. Gel preparation for SDS PAGE for purified protein samples In order to obtain improved resolution of protein bands, samples were run on 520% gradient gels. This refers to the amount of acrylamide present in the gel matrix; there being 5% at the top of the gel and increasing to 20% at the bottom. Two gel solutions were prepared. The “weak gel”: Tris HCl, 3 M, pH 8.8 4 mL (72.66 g Tris in 200 mL water, buffered to pH 8.8) 87 Chapter 2 Materials and Methods SDS (10% w/v) 300 µl Acrylamide mix 5 mL (30 mL BioRad 40% (w/v) acrylamide/bis solution + 10 mL water) Water 20 mL The “strong gel” Tris HCl 3 M, pH 8.8 4 mL SDS (10% w/v) 300 µl Acrylamide mix 20 mL Water 3.2 mL Sucrose 2.4 g Both solutions were kept on ice and there was enough to pour two gels. Two sets of 205 mm x 205 mm glass plates (one long plate and one short plate with a gap at the top end) were cleaned with alcohol and water and placed in the gel former, with 1mm spacers at each side, and tightened. A gradient former was used to mix the gel solutions in order to create the acrylamide gradient across the gel. The gradient former was placed on a magnetic stirrer, a stirrer bar was placed in both chambers and a length of tubing connected to the outlet, this lead to the gel former passing through a peristaltic pump. A yellow pipette tip was placed on the end of the tube to allow the gel mixture to run down between the glass plates. Both outlets on the gradient former were closed, 13 mL of weak gel was placed in the left hand chamber, the connection between the chambers was briefly opened to allow a small amount of gel to flow into the right chamber in order to avoid an airlock. Strong gel (13 mL) was filled into the right chamber. To both chambers 300 μL ammonium persulphate (75 mg/5 mL) and 7 μL TEMED (N,N,N’,N’-tetramethylethylenediamine) were added and mixed using a pipette tip. The connecting outlet was opened, the magnetic stirrer and the pump switched on and the outlet to the gel former opened. This caused the gel to be filled; the flow of weak gel from the left chamber diluted the gel in the right chamber causing an even reduction of acrylamide concentration throughout the gel. After pouring the gel, water was placed on top to ensure an even surface at the top end of the gel. After pouring the second gel, both gels were covered with clingfilm and stored overnight at 4ºC. The following day the water was poured off the gels and the space on top of the gels was dried with pieces of filter paper. Care was taken not to touch the surface of the gel. The stacking gel was prepared as below (enough for 1 gel). Water 3.4 mL Acrylamide Mix 630 µL Tris HCl 0.5 M, pH 6.8 880 µL 88 Chapter 2 Materials and Methods SDS (10% w/v) 50 µL Ammonium persulphate 50 µL (50 mg/450 μl water) TEMED 10 µL This was mixed and poured on top of the resolving gel using a Pasteur pipette. An 18 well comb was inserted into the stacking gel, care being taken to create no bubbles. The gels were removed from the former and inserted into the electrophoresis equipment (Flowgen VM2020-DW) The running buffer was prepared as below. Running buffer (5x concentrated) 240 mL (Tris 15 g, glycine 72 g in 1000 mL water) Water 960 mL SDS 1.2 g This was poured into the top of the equipment between the gels and into the low sections at the base of the two gels. During electrophoresis the system was cooled with tap water. The combs were removed from the gels. The samples were prepared and centrifuged in a microfuge (Quick Fit Instrumentation) for two min to remove any particles that could cause streaking on the gels. The samples and the molecular weight standard were loaded using gel loading tips. The system was connected to a power supply (BioRad 200/2.0 Power Supply) and the gels run at 200 V for four h. 2.33.1 Silver staining of SDS-PAGE gels The gels were stained in large glass trays that had been washed with nitric acid (approximately 50% v/v) and rinsed with distilled water. After electrophoresis the plates containing the gels were removed from the equipment and placed in the glass tray with the plates leaning against one end of the tray. The short plate was prised off leaving the gel on the long plate. This was rolled off using a clean spatula, care being taken not to touch the gel by hand. The long plate was removed along with the stacking gel. The gels were treated with a series of solutions. The trays were placed on shaking platforms (Stuart Scientific STR6) set a 20 rpm. The gels were immersed in a fixative solution (see below). 89 Chapter 2 Materials and Methods Methanol 800 mL Acetic Acid 160 mL Water 640 mL This was enough for both gels. The solution was left for 30 min then removed using a Pasteur pipette tip attached to a Buchner tap attachment. This was followed by a second fixative solution: Methanol 80 mL Acetic Acid 120 mL Water 1,400 mL This solution was removed after 30 min and the gels were washed for one h in water that was changed twice. The next solution was 8 mg dithiothreitol in 1,600 mL water for 30 min, followed by 1.6 g silver nitrate in 1,600 mL water for 30 min. The developing solution contained 60 g sodium carbonate in 2 L water with 1 mL formaldehyde (40% v/v). Each gel was briefly washed in water, washed with a few mL of developing solution, and then developed in 600 mL developing solution. The development was stopped by addition of 30 mL of 2.3 M citric acid (48.332 g/100 mL). This was left to mix for 10 min and the gels were washed twice with water. The gels were preserved by sealing them into plastic pockets using a thermal sealer and stored at 4ºC. Polyacrylamide gels form after polymerisation of monomeric acrylamide into polymeric acrylamide chains and cross linking of the chains by N,N’- methylene bisacrylamide. Polymerisation is initiated by addition of ammonium persulphate and accelerated by TEMED by forming free radicals. Mercaptoethanol is added to the sample buffer as this denatures proteins when heated. The SDS (sodium dodecyl sulphate) in the buffer ensures that the denatured proteins all have identical charge densities so that the proteins migrate through the gel according to their size and not charge. Bromophenol blue was added as a tracking dye to locate the samples in the gel. Silver staining works by reduction of ionic silver to metallic silver. Detection of protein in a gel requires difference in the oxidation-reduction potential between the sites occupied by proteins and the rest of the gel. If a protein has a higher reducing potential than the gel, then the protein will stain brown/black, if it has a lower potential, the protein will stain negatively (white). Dithiothreitol was included in order to maintain the proteins in a reduced state. 90 Chapter 2 Materials and Methods 91