Characterisation of Bovine Serum, Haemoglobulin, Albumin and Immunoglobin using SDS-Page.

Abstract:

Known molecular markers, (phosphorylase B, Bovine serum albumin, Ovalbumin, Carbonic

anhydrase and lysozyme) were run on a SDS-Page electrophoresis gel alongside bovine serum,
bovine serum albumin, (BSA), haemoglobin (Hb), globulin fraction, and albumin in their respective
wells. All the samples were treated with Sodium Dodecyl Sulphate and bromophenol at 100oC.A
two gel systems was used which served to increase resolution as the stacking gel ensured that the
proteins started migrating at the same point by concentrating the loaded samples into a single thin
band. Upon reaching the 10% polyacrylamide the BS,(L1);BSA(L2);Hb(L3);Globulin
fraction(L4);albumin fractio(L5);and the known molecular markers separated into various
components in order of their respective sizes characterised by bands upon staining with comassie
blue. BS showed two distinct bands of sizes 67kDa and 52kDa; the BSA a 59kDa band, whilst Hb
on the other hand two also showed bands of 46 kDa and 12,5 kDa (L3); Globulin fraction shows
two bands with respective sizes of 46 and 13,7kDa.(L4); albumin fraction has one major component
of 46kDa.

Introduction:

Opposite charges attract, so much so that if an ion,(charged particle),is placed across a electric field
it will migrate to the electrode with opposite charge to itself, this is know as electrophoresis. The
migration of ions is described by White as been dependant of a numerous factors that are unique to
the species in question, such as wetted area, friction and size, just to mention a few).

In biochemistry and other sciences various electrophoresis techniques, (iso-electric focusing,sds-

page,2D electrophoresis) have been employed as a qualitative and quantities tool as early as the
1900's,where samples were allowed to migrate on a support medium such as paper, polyacrylamide,
agarose, etc. Polyacrylamide2 gels are made by polymerisation of acrylamide and bis-acrylamide in
the presence of free radical forming agents such as ammonium per sulphate or riboflavin, in the
presence of N,N,N'N'-tetramethylethylenediamine,(TEMED).

A variety of isolation and characterisation methods have been developed based on electrophoresis
with each method having its own merit in terms of resolution, reproductively and ease of
application. Take isoelectric focusing (IEF) for example, it can separate charged species to a degree
of a minus one charge, (phosphate), yet it does very little for mass characterisation. Whilst SDS-
Page on the other hand separates the protein components based on their size, by initially treating all
the protein samples with a boiling anionic detergent,(sodium dodecyl sulphate),we ensure that they
all have a negative charge proportional to their respective sizes and that they are all the same
shape(rod-like)3.Placing these proteins in electric field with polyacrylamide support medium we are
assured that they will migrate toward the cathode,(positive electrode) and more importantly they
separate solely based on their size and they have lost all other uniqueness besides their charge they
carry which has a direct correlation to size.

The treated proteins forms rods of different sizes, all with different charge, the charge primarily
pulls the proteins towards the cathode but as it migrates through the gel matrix its motion is
impeded by the cross linkages of the gel. The protein will continue to move towards the cathode
until it reaches a point were the Electric field strength felt by the protein due to its charge is equal to

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the frictional force imposed by the gel cross linkage. All proteins of the same size will be
concetrated within the same vicinity and form a band which can be seen and quantified after
staining with a suitable dye, (comassie, amido black, silver, etc.)1. Biomolecules such as proteins
are very unique in structure and so upon denaturation will form unique sub-units which during a
SDS-page will travel through different distances depending on their sizes.

SDS-page offers a few advantages, it is relatively easy to setup and run a SDS-page experiment ;
SDS detergent solubilises most proteins, and due to its anionic nature ensures high mobility of
treated samples, this indirectly prevents running electrophoretic gels for long times, thence heating-
up of gels. Distance migrated by proteins through polyacrylamide gels show a negative inverse
relationship and a plot of distance against Log M of a known molecular marker one can work out
sizes of unknown proteins. Disadvantages include the toxicity of the reagents used for preparing the
gel slab, treating proteins with SDS denatures them, also SDS does not bind very well to
glycoproteins so yields inconsistent results.

In this report we describe the application of SDS-Page for qualitative and quantitative analyses of
separated and isolated components of Bovine Serum, ( BS). Known molecular markers were run
alongside parallel SDS-Page electrophoresis wells containing bovine serum, bovine serum albumin,
(BSA);haemoglobin (Hb);globulin fraction ;and albumin. After running samples through gel it was
stained with comassie blue, viewed under black light from which the protein bound Bromophenol
glows. Measuring the distance travelled by the various known components of the molecular marker
and plotting them against their Log molecular weights were able to get a calibration curve from
which sizes of the other samples could be calculated. Judging the intensity of bands characteristic of
the different proteins enabled us to comment on the level of concentration of the isolated BSA
fractions.

Methods:

Refer to practical manual

Results:

Samples of known molecular markers; bovine serum, bovine serum

albumin,(BSA);haemoglobin(Hb);globulin fraction ;and albumin were all treated with SDS at
100oC ,and then with bromophenol prior to being loaded to stacking gel in the two gel SDS-page
system. Using a two gel system served to increase resolution as the stacking gel ensures that the
proteins start migrating at the same point by concentrating the proteins into a single thin band. Upon
reaching the 10% polyacrylamide the BS,(L1);BSA(L2);Hb(L3);Globulin fraction(L4);albumin
fractio(L5);and the known molecular markers separated into various components in order of their
respective sizes ,characterised by bands upon staining. After running samples through gel it was
then stained with comassie blue,

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Figure 1.BS has two distinct bands of sizes 67 003Da and 52 240Da; the BSA has a 59 170 Da
band, whilst Hb on the other hand shows two distinct bands of 46 142 Da and 12,5 kDa respectively
(L3); Globulin fraction shows two bands with respective sizes of 46 and 13,7kDa.(L4); albumin
fraction has one major component of 46 142Da.

Plotting distance travelled by molecular marker proteins against their relative molecular weights to
give a calibration curve, (figure 1).

Figure 2.Plot of distance migrated by

molecular markers (M) on a SDS-page
against their log molecular weight,
afforded a calibration curve with a
negative linear relationship of the
equation y = - 0.027x + 5.3391.

Discussion:

Bovine serum (L1) has proteins with six different sizes, with the first band,(67kDa) having the
highest intensity, as there is a direct relationship between intensity of band and protein
concentration it can be concluded that it is the major component of the Bovine serum. Cross
referencing this band with lanes 2 and 5 led to the conclusion that it is in fact albumin. The proteins
characteristic of band 2 and 6 respectively of lane 1, have molecular masses of 52 240Da and 13
308Da.These two bands compare favourably with those bands characteristic of lane 4's globular
protein. Bands 3, 4 and 5 have proteins of masses ranging from 19-33kDa respectively, these are
smaller components of Bovine serum, (hormones, growth factors, etc).

Lane 2 (BSA) has four visible bands the most distinct band is 59 kDa, which is the major protein of
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bovine albumin. The array of bands is evident of the fact that albumin is not just a single protein as
is generally believed but rather a mixture of proteins all having similar properties.

Globulins (L4) are said to be proteins that are part of serum but not as soluble as albumin, a very
common example of such proteins are immunoglobulin. Immunoglobulins are Y-shaped
symmetrical molecules with four sub-units,(two light chains(23kDa each) and two heavy
chains(53kDa each)).One half of the molecule is made up of a heavy chain bound to a light chain,
these half’s are then joined in the centre by disulphide bonds to constitute a immunoglobulin. When
performing a SDS-page we attempt to make the protein molecules as uniform as possible, part of
this entails treating the samples not only with SDS but also beta mercaptoethanol which serves to
break any disulphide bonds between the proteins. More so in this case we expect this to be Ig as it is
the major antibody found in mammals and it is the only antibody with the ability to cross the
placenta into the bovine foetus from which BSA is collected. The globulin fraction from the ion
exchange chromatography has separated into two bands, (14 and 46kDa),these bands are most
probably the light and heavy chains respectively.

Albumin, (Lane 5) has a chromatographic profile similar to that of the BSA(lane 2). It too has a
major band at 59kDa and differs in that it is more pronounced than lane 2,due to it having a higher
concentration of albumin.

Haemoglobin2 (68kDa) is a tetramer protein with four identical subunits each of 17kDa.Running Hb
(L3) on a SDS-page shows two major bands at 59 and 12.5 kDa. The 12.5kDa band is the most
distinct and is representative of the individual subunits that resulted from Hb being denatured by the
SDS; the second heavier second band represents Hb that is still in tact, (i.e. is still in its native
state).

Profiles of samples in lanes 2, 3, 4, and 5, (BSA; Hb; globulin fraction; and albumin, respectively),
are fragments of a more comprehensive SDS-page band profile of Lane 1, (BS).

Though SDS-page is easy to carry out it fails tremendously it is not a very accurate method of
determining protein sizes, as the calibration curve is a plot of Logarithm very small errors on the gel
are exaggerated when applying the curve. This is seen in our experiment where all the Mw values of
the calculated proteins are slightly off from the literature values.

References:

1. Robyt, J.F and White B.J (1990) Biochemical Techniques Theory and Practice. 129-142

2. Mathews, C.K., van Holde K.E. and Ahern K.G. (2000) Biochemistry 3rd Edition. 591-598

3. Dryer,R.L and Lata G.F (1989) Experimental Biochemistry. 128 – 129

4. http://en.wikipedia.org/wiki/albumin

5. http://en.wikipedia.org/wiki/serum

6. http://en.wikipedia.org/wiki/immunoglobulins

7. http://en.wikipedia.org/wiki/haemoglobin

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