SDS-PAGE WITH DISCONTINUOUS BUFFER SYSTEM OF GOAT

Pakistan Vet. J., 18 (4): 1998
SDS-PAGE WITH DISCONTINUOUS BUFFER SYSTEM OF GOAT
MILK WHEY
Munazza Shauket, M. Ashfaque, I. Hussain and T. M. Chaudhry
Department of Veterinary Microbiology, Faculty of Veterinary Science,
University of Agriculture, Faisalabad, Pakistan
ABSTRACT
Polyacrylamide gel electrophoresis is a valuable tool for establishing quantitative distribution of milk
proteins. The fat free goat milk was obtained by centrifugation. The casein was removed by coagulation with
2 per cent solution of rennin. The whey was purified by filtration and then it was subjected to 12.5 per cent
polyacrylamide gel electorphoresis. Six protein markers, bovine serum albumin (dimer), bovine serum
albumin (monomer), chicken egg albumin, carbonic anhydrase, haemoglobin and lysozyme were also run
in the same way. Two out of fi've goat milk whey samples, presented five protein bands including a
lactalbumin, H-lactoglobulin, lactoferrin, serum albumin and unidentified protein with Rf values ranging
from 0.34 to 0.72 having molecular weights ranging from 14.3 kDa to 87 kDa. The other three samples had
a similar protein pattern except that the protein band with Rf value 0.593 having molecular weight of 30 kDa
present in two samples was absent.
distribution of milk proteins. Five distinct milk proteins
INTRODUCTION
by electrophoresis are serum albumin, B-lactoglobulin,
a-lactalbumin, pseudoglobulin and euglobulin (Pearce
Electrophoresis is a widely used chromatographic
technique for the separation of mixutres of ionic
and Shanley, 1981). The objective of the study was to
compounds. Zonal, or gel electrophoresis combines
determine the goat milk whey proteins by a suitable,
elements of free boundary electrophoresis (separation
modern and reliable techniqut! to know whether it could
based on charge) and gel filtration (separation based on
be used as a foetal calf serum substitute for cell culture
size). Gel electrophoresis has been adapted extensively
studies
or
not.
This
paper
describes
only
the
as a tool in preparative and analytical biochemistry. Disk
determination of goat milk whey proteins by SDS
gel electrophoresis containing sodium dodecyl sulphate
PAGE.
(SDS) provides improved resolution over a continuous
MATERIALS AND METHODS
buffer system. Discontinuities in buffer composition,
pour
size
and
pH
produce
an
isotachophoretic
Purification of \Vhey
concentration of the sample in a low percent acrylamide
stacking gel, before the size separation in the resolving
Whey was separated and purified by following the
gel. Because the sample is concentrated into a band of
technique described by Akhtar er al. (1992). Briefly five
only micron wide before separation, the tinal degree of
fresh goat milk samples were collected and were made
diffusion is greatly decreased. Even large amount of
fat free by centrifugation at 3,000 rpm . for 20-25
sample can be applied without reduction of resolution.
minutes. Rennin (2%) at rate of 0.01 ml/ml of milk was
As in the continuous buffer system, the density of the
added in fat free milk to separate the casein. The sample
gel net work of the resolving gel varies with the
was incubated at 37°C till curdling. The whey was
molecular weight range of the samples. In addition to
separated from clotted milk by centrifugation at 2,000
this, buffer concentrations and pH values also varies to
rpm for 20 minutes. The whey thus separated was
provide the concentrated effect in the stacking gel and
filtered
the maximum possible resolution in the resolving gel.
subsequently through membrane filter of 0.22.um pore
through
Seitz
filter
assembly
and
then
size. The purified whey obtained in this way was
Polyacrylamide gel dectrophoresis (PAGE) is a
subjected to 12.5 per cent (SDS-PAGE).
valuable tool for establishing qualitative and quantitative
173
Pakistan Vet. J., 18 (4): 1998
174
The other three samples had a similar protein pattern
SDS-PAGE
Discontinuous buffer system of. SDS-PAGE as
described by Laenm1li ( 1970) was used. Separating gel
buffer ( 1.5 M tris HCl, pH 8.8), stacking gel buffer (0.5
tris HCI, pH 6.8), 2X sample buffer (tor liquid sample).
IX sample buffer (for solid samples) and runnin buffer
(electrode buffer ) were prepared following the method
described by See and Jackowsky ( 1989) and stored at
4c'C. Vertical gel ele�trophoresis system (BIORAD
USA ) was used for the separation of whey proteins.
The samples were prepared by adding 50�' L of 2X
samples buffer to 50ML of protein �mpel (purified
whey ) in 1.5 mL eppendorf tuhes. A 3�,L of 0.2 per cent
bromophenol blue was added and kept in water bath at
lx>iling temperatur� tor two minutes and cooled at room
temperature. Six protein m�arkers of de\.:trophoresis
grade were selected and m�arked "M" on eppendorf
tubes; i.e., M-1: Lysozyme, M-2:Heamoglobin, M-3:
Carbonic anhydrase, M-4: Chicken egg albumin, M-5:
Boving serum albumin (Monomer) and M-6: Bovine
serum albumin (Dimer). Protein m.arker (2 mg) was
.. mixed with 200.uL of I X sample buffer in eppendorf
tube marked "M ". A )Li L of 0.2 percent bromophenol
blue was added to each tube and was kept in water h�Lth
at boiling temperature for two minutes and the cooled at
room temperature.
The samples were loaded (20 J!L) in to the gel slots.
Electrophoresis was carried out at room tem penat ure �at
100 V h>r 6 hours until the bromophenol blue dye wus
a bout 1 em from t he bottom of separation gd. After that,
the gel w;.as su�jecteJ to st a ining and dest�tining. (Hames
an d RickwooJ, 1983).
Molecular weights were d etermi n ed as describeJ by
Weber <tnd Osborn ( 1969) from rekative mob il ity (Rt) by
extrapolating from a st�md:.trd curve of molecular weight
except that the protein band with Rf value 0.593 having
molecular weight of 30 kDa present in two samples
described above was absent. There was a visible
difference in concentration of proteins in all five milk
whey samples in respect to intensity of staining of
individual hand (Plate 1 ).
87.0KDa
67.0KDa
30.0KDa
19.0K.Da
14.3KDa
Phate I.
Table 1:
Lane
No.
2
markers as shown in Fig I .
RESULTS
In SDS-PAGE using 12.5 per cent polyacrylamide
gel, two out of tive goat milk samples, presented tive
protein bands with Rf values ranging from 0.34 to 0.72
having molecular weights ranging from 14.3 to 87 kDa
(Table I). Protein band with Rf value 0.340 had
molecular weight of 87 kDa and that of Rf value 0.373
had molecular weight of 67 kDa. Protein band of Rf
value 0.593 had molecular weight of 30 kDa. Protein
band Qt'Rf value 0.70 1 had molecular weight of 19 kD:l.
Protein band of Rf value 0.72 h�td molecular weight of
14. 3 kDa.
SDS-PAGE of �llut milk whey using 12.5%
polyucrylmniJ gel.
3
Molecular weight of protein bands of goat
milk whey with their Rf values
No. of
B�mds
Molecular
Wei!!ht (kDa)
2
0.340
0.373
87 . 0
67.0
3
0.593
30.0
4
5
1
0.701
0.720
0 340
19.0
14. 3
87.0
2
3
4
0.373
0. 70 1
0.72 0
67 0
1
2
3
0.340
4
5
.
0.373
0.593
0.70 1
0.7 20
0.340
4
2
3
5
Rf value
4
I
2
3
4
0.373
0. 70 1
0.720
.
19.0
1 4. 3
87.0
67 .0
30.0
19.0
14.3
87.0
67.0
0.340
19.0
1 4. 3
87.0
0.373
0.70 1
0.720
67.0
19.0
1 4. 3
175
1998
acid, the re sulting whey contains the soluble p roteins
Out of ti ve milk samples, three had four protein
similar
(4):
Larson (1985), when casein is precip itated from milk by
DISCUSSION
fnlctions. Almost
Pakisum Vet. 1., 18
o bservation s
have
b een
reported by Stupnitskii and Chenko {1967) that goat
incl uding the proteose peptone fragments der ived by the
c l eavage of H-casein as casein was removed by rennin.
The protein bands with molecular weight of 1 9. 0 kDa
milk whey proteins were separated by gd elect rophoresis
was present in all tive milk whey samples resembled the
( lgs), blood se rum albumin (BSA), a -lw. : talbu min (a
( 1985) and S higeru (1988) observed the molecular
into four fraction s correspo n ding to immun oglobulins
usin g
B-lactoglobu li n as 18.3 kDa. The protein
bands having molec ular weight of 14.3 kDa resembh!d
in molecular weight of ex-lactalbumin (a-LA). Larson
The protein fractions with mo lec u l ar \veigh t of 87
as 14.17 kDa, where as Shigeru ( 1988) o bserv ed the
LG). Cossedu and Pisanu (1979), Lim eta/. ( 1986) anJ
Nhllua n e1 a/. ( 1988) also rep or t e d the presen�:e of four
fntdions
protein
in
goat
milk
whey
polyacr yhuniJ e gd electrophoresis.
by
kDa present in all smnples of goat milk whey resemhleJ
in molecular weight of hH.: to ferrin. S h ig er u ( 1 9 8 8 ) also
repor teJ the molecular we i ght of lactof�rrin as 87 kDa,
while Lurson (1985) Jescrihed the molecular weight of
lactot�rrin as 90 kDa. Lactot�rrin is an iron binding
protein present in milk. Transferrin is also an iron
binding protein which is common in bloo d pl asm a.
Lactoferrin is secreted by several other organs besid es
th e_ mammury glands. Both these differ from e �tch other
in compo s i t i on
.
and electro phoretic mobility and do
not
Q
;:
ce
'5
l.i
Q
0
E
C)
.Q
weight of
( 1985) described the molecular weight of a-lactalbumin
mol e cu lar weig h t of
a-lactalbumin as 14.2 kDa a
- lac ta lb umin exerts a protective ad ion on the large
mo l ecu �d r a gg regat i o n s of milk, su c h as casein and fat.
globules. Lactalbumin because of its hig h lysi ne content
also exerts a desi ra ble supple mentary e ftt!ct i n impro v ing
the nutiritive value of casein. It also plays an essential
role in
the bios ynth esis of lactose.
The vari ;;t tion
observed in molecular weights of protein fractions exists
in literature too.
Th e i ntensity of protein banJs was dift�rent in all
g oat milk wh e y sam ples.
Ce rta i n phys iolog i c al and
p�lthologic;.d fac t ors int1uence the concentration of
prot e ins in milk. A1.:�oruing to the tindings of Birgel e1
( 1971 ) . the amount of y-globulin rises in milk with
�
51
E
.2'
molecular weight of B -lactoglob uli n (H-LG). Larson
a/.
49
age while albumin anJ H - glob ulin are not i n fl u en ced by
4 �
age.
4-
Cossedu
a nd
Pisanu
( 1979)
obse rved
that
immuno glob ul i n content of goat w hey proteins was
4 ;,
higher in
.:5
.:.:
Fchruary
lactglo bulin anJ
43
and
lower
P -la ctalbumi n
in
June while H
contents were lower in
Februar y than in o th er mo nths . R<linar d el al. ( 1982)
42
reported th at transferr in and bov ine seru m al b umi n
4,
4
0
01
02
�
J
:
.:
05
06
07
08
ss
inc reased su fficiently in late laL'tation (270th day of
la c ta ti o n) .
Lactoferrin
concentration
i nc reas ed
si g ni tic a ntly in q uarter s infected by major pathogens
Fig.l:
S tanda rd c urve of pro te in markers
cross react \Vith each other immunologically . Transferrin
hus bee n isolated from blood of many spec ies but only
from the milk of cow and rabbit (Smith, 1959).
Lactot�rrin has been isolated from milk of h umm1s,
mouse, guinea pig, cow an d shee p . The p rotein fractions
with molecul•lr weigh t of 67 kDa present in goat milk
whereas m i nor p ath ogen s infection caused no signiticant
mcrease in lactoferrin c on cen tratio n ( Rainurd et al.,
1982).
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