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
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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
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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.
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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 25C and 37C (Figure 2.1).
Lager strains grew at 25C and not at 37C, 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 25C 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.
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Chapter 2
Materials and Methods
Ale yeast
strain
25C
37C
Figure 2.1. Growth of lager and ale yeast strains on peptone-yeast extract glucose nutrient agar plates for
48 h at 25C and 37C.
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).
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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 his31 leu20
met150 ura30. 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.
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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.
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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
11C 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 65C for 1 h and the temperature raised to 74C 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 12C for lager
fermentations and 20C for ale fermentations. The conditioning temperature was 5C for
lagers and 9C for ales and in both cases the temperature was adjusted to -1.5C 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
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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 18C for
the 10 Plato and 14 Plato fermentations and 20C for the 18 Plato fermentation.
Conditioning temperature was maintained at 5C and the temperature adjusted to -1.5C
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
10C
10C
14C
Lager yeast strain
Strain A
Strain A
Strain A
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Fermentation temp
18.5C
18.5C
20.0C
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 20C 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 20C was expressed as the
density relative to water at a temperature of 20C (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 20C relative to distilled water
at 20C.
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
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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 20C 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
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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
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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
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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
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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
20C 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
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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,
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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 30C for 30 min. The reaction was
terminated by heating at 80C 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
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(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
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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.
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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.
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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).
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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.
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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.
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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.
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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
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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
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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).
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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.
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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.
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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
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(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
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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
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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
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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
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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)
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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
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Chapter 2
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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).
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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.
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91