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Post-laboratory Report on

Exercise 4.4
Gel Electrophoresis

Vikki Anne R. Cedo


CHEM 160.1 - 3L
2nd Semester 2014-2015

Groupmates:
Desiree Joy Cerico
Ma. Kriselle Ornales
Mary Ranzelle Pasang

Date performed: March 17, 2015


Date finished: March 18, 2015

Ms. Korina Vida G. Sinad


Laboratory Instructor

Exercise 4.4

Gel Electrophoresis
Powerful electrophoretic techniques have been developed to separate
macromolecules on the basis of their molecular weight. The mobility of a molecule
in an electric field is inversely proportional to the molecular friction which is the
result of its molecular size and shape; directly proportional to the voltage and
charge of the molecule. Gel electrophoresis is a technique wherein molecules are
separated on the basis of their molecular weight and charge. Various gels can be
used in this technique such as polyacrylamide and agarose.
Polyacrylamide gel electrophoresis or PAGE separates polypeptides according
to their molecular weights. Proteins that are negatively charged by the binding of
the anionic detergent sodium dodecyl sulfate separate within a matrix of
polyacrylamide gel in an electric field according to their molecular weights.
Table 4.4.1. IpH's and direction of migration of proteins.
Protein

IpH

pH

Pepsin
Lysozyme
Hemoglobin
Myoglobin

1.0
11.0
6.8
7.0

5.0
7.0
7.0
5.0

Direction of
migration
Anode
Cathode
Anode
cathode

Co-polymerization of acrylamide and bis-acrylamide form the polymerization


gels. The reaction is a vinyl addition polymerization initiated by a free radicalgenerating system (Chrambach, 19850). Ammonium persulfate is added to the gel
recipe / gel mixture in order to initiate the gel polymerization by activating TEMED
or tetra methyl-ethylenediamine. Polymerization is initiated by TEMED and
ammonium persulfate. Ammonium persulfate yields a persulfate free radical which,
in turn, activates TEMED. When activated, TEMED acts as an electron carrier to
activate the acrylamide monomer into a free radical state. This activated monomer
then reacts with an unactivated monomer to begin the polymer chain elongation
(Shi and Jackowski, 1998). Bis-acrylamide randomly crosslinks the elongating
random polymer chains, which then results in closed loops and a complex "web"
polymer with characteristic porosity that depends on polymerization conditions and
the monomer concentrations.
Migration of molecules, in general, are faster with higher voltage.
Migration and voltage have a linear relationship. Acrylamide concentration, as well
as bis-acrylamide concentration (cross-linker), determines the relative size of the
pores that will be formed in the gel. As the total amount of acrylamide increases,
the pore size decreases. We can say that the concentration of acrylamide and the
pore size is inversely proportional. 5% of the cross-linker gives the smallest pore
size with cross-linking. Any increase or decrease in the amount or concentration of
the cross-linker will result to the increase in pore size The addition of beta-

mercaptoethanol on the gel increases the resolving power of the gel. Betamercaptoethanol is a reducing agent used in breaking disulfide bonds, thus it
ensures that the protein is fully denatured into its sub-units; because proteins need
to be linear in order for the PAGE to proceed properly.
Increasing the temperature to a very high level will cause the gel to melt.
Temperature affects the properties of the gel (Chen and Chrambach, 1979).
polymerization at 04C results in turbid, porous, inelastic gels, and reproducibility
is difficult to achieve. These properties may be due to increased hydrogen bonding
of monomer at low temperatures. Also, the kinetic energy of the molecules
increases when the temperature is increased resulting to higher rate of migration.
Table 4.4.2. Molecular weight or sample and standards and their Rf values.
Protein
standards

Lane
no.

MW
(kDa)

Distanc
e
travele
d by
dye
7

500
350
250

Distance
traveled
by
sample
(cm)
2.3
3.2
3.1

Rf

Log MW

Egg albumin
extract (crude)
Ammonium
sulphate
fraction (60%)
Fraction 9 of
Gel chrom.
Albumin isolate
Fraction 10 of
Gel chrom.
Albumin isolate
Fraction 11 of
Gel chrom.
Albumin isolate

0.32
0.46
0.44

2.70
2.54
2.40

90

3.3

0.47

1.95

70

3.4

0.49

1.85

50

3.5

0.5

1.70

Calculations:
Rf = distance travelled by sample / distance travelled by tracking dye
Example: for egg albumin extract
3.2 / 7 = 0.46

3
2.5
2
Log MW of samples and standards

1.5
1
0.5
0
0.43 0.44 0.45 0.46 0.47 0.48 0.49 0.5 0.51
Rf values

R = -0.98

Slope: -11.1904

R2 = 0.965

Linear equation: (-11.1904x) + 7.29

y-intercept: 7.29
Calculations:
by interpolation,
log MW = (-11.1904x)(Rf value) + 7.29
Example: Egg white albumin: log MW = (-11.1904x)(0.46) + 7.29 = 2.1424
MW = antilog (2.1424) = 138.80
Due to errors in performing the experiment, estimating the concentrations of
the samples in the gel, and getting the Rf values, the computed molecular weight is
not anywhere near the theoretical value or standard values. The acceptable linear
coefficient is 0.99-1. In this experiment we obtained a negative linear coefficient,
thus not achieving the experiment's objective.

Figure 4.4.1. Gel electrophoretogram visualization of proteins.

Native PAGE is a method commonly used to separate native proteins. The


conditions of the setup are set so that the proteins that are migrating in the gel are
kept in their native state. The buffers used in native PAGE proveide a nondenaturing, native-like milieu; the electrophoresis is performed at low temperature
so the heat can dissipate. Numerous enzymes retain native conformation and
enzymatic activities (biological function) while running in the gel. Native PAGE is
also a useful method for checking the uniformity of an isolated protein. Even if the
purified protein sample contains only a single type of protein, the sample might not
be uniform such that some of the molecules might be unfolded or has undergone
some chemical modifications. Unfolding of proteins change the overall shape of the
molecule; most chemical modifications change the electric charge of native
molecules. If no side products or contaminants are present in the sample, it is
expected to be visualized as a single sharp band, otherwise, smearing of the band is
expected. In addition, native PAGE can also be used in the detection of complex
formation between proteins. If a complex is present between two or more proteins
(or proteins and non-protein ligands), the complex can be detected as an extra band
in the gel. This is due to the fact that in native-like conditions, many non-covalent
interactions are maintained and the complex migrates apparently as a single
molecule.
SDS PAGE or sodium dodecyl sulfate polyacrylamide gel electrophoresis is a
method used to separate proteins. However, unlike native PAGE, the proteins in SDS
PAGE migrate in their denatured state. The migration velocity of proteins is a
function of their size, shape and number of electric charges they carry. As the

velocity is a complex function of these properties, native PAGE cannot be used in


estimating the molecular mass of proteins. The traditional native PAGE is similarly
unable to assess whether a purified protein is composed of a single subunit or
multiple subunits. The SDS gel separates individual polypeptide chains (monomeric
proteins and subunits of multimeric proteins) according to their size. SDS is an
anionic detergent. Proteins treated with SDS at high temperature show radical
conformational changes. This breaks all native non-covalent intermolecular (intersubunit) and intramolecular interactions. Multi-subunit proteins' subunit structure
disintegrates and the proteins unfold. If native conformation is stabilized by
disulfide bridges, reducing agents are added to open up connections. SDS molecules
bind to unfolded proteins in large excess, providing extra negative charges to the
molecules.
SDS PAGE is a standard method for assessing the homogeneity of an isolated
protein. It is also a robust method for analysis of supramolecular complexes like
multi-enzyme complexes. SDS PAGE separates and denatures individual subunits of
these complexes. Thus making the polypeptide chains migrate separately in the gel.
Various staining procedures can be used to visualize all subunits and relative
amounts of these subunits can be determined or estimated. This allows
identification of each subunit of a complex.
Both gel filtration and electrophoresis are used as a separation technique,
their difference is that when gel filtration is used larger molecules elutes first and it
is non- denaturing while in electrophoresis smaller molecules elute first and it is
denaturing. Gel chromatography can be used for desalting, MW determination and
purification whereas electrophoresis is used for purification, subunits determination
and MW determination.
The advantage of using electrophoresis is that the gels are easy to prepare
and that samples can be loaded and run simultaneously. Electrophoresis has a very
high resolving power. Its disadvantage would be that electrophoresis requires the
sample to be denatured and that the equipment is expensive. It also requires
electricity and the use of acrylamide, a neurotoxin; so you have to be very careful in
handling the chemicals and reagents when preparing the gel.
Gel filtration chromatography, the movement of the molecules is only based
on the gravitational pull; because you will just wait for the molecules to elute out of
the gel. It only requires simple equipment in order to be performed and the protein
samples do not have to be denatured in the process, keeping intact their native
conformation and biological activity. The disadvantages of the technique include: it
is time consuming and more effort is needed because of the preparation of the
sieving column, collection of fractions is done one-by-one and this takes a lot of
time, must be monitored from time to time and the resolving power is not effective
enough to separate very slight differences in MW.

Literature Cited
Bettelheim, F. (2007). Introduction to General, Organic and Biochemistry.
Brooks/Cole by Thomson Learning.
Chen, B. and Chrambach, A., (1979). Estimation of polymerization efficiency in the
formation of polyacrylamide gel, using continuous optical scanning during
polymerization, J Biochemistry and Biophysics Methods 1, 105-116
Chrambach, A., (1985). The Practice of Quantitative Gel Electrophoresis, VCH,
Deerfield Beach.
Shi, Q. and Jackowski, G., (1998). One dimensional polyacrylamide gel
electrophoresis, pp 1-52 in Harnes BD (ed) Gel Electrophoresis of Proteins: A
Practical Approach, 3rd edition, Oxford University Press, Oxford.
Wang, N. S. (n.d.). Chemical & Biomolecular Engineering Official Site. Retrieved
March 2, 2015, from Maryland University Department of Chemical & Biomolecular
Engineering: http://www.eng.umd.edu/~nsw/ench485/lab6c.htm