NAME: Merve MURAT 05.04.

2017
ID: 2004141
SECTION / GROUP: 3 / 1
SUBMITTED TO Ouzhan BEK

SDS-PAGE

ABSTRACT
SDS-PAGE was used to determine the molecular weight of -chemotrypsin, BSA, Egg-
albumin in this experiment and to compare with total protein data. In addition, it can be
mainly used for determination of protein purity and comparison of protein concentration.
Briefly, this method includes denaturation and linearization of proteins by using chemical
reagent, and separation of them with respect to their size under uniform electric field
(electrophoresis). Protein functions and covalent bonds are firstly destroyed, then all proteins
are possessed of the same charge; that is, all of them has negative charge. In electrophoresis
part, proteins are separated and they forms bands on polyacrylamide gel after staining
procedure. The molecular weight of proteins can be determined by comparison with ladder
bands.

INTRODUCTION
Secreted proteins or extracellular (cell surface) proteins must be stabilized enthalpically and
must be folded by the help of covalent interactions and mainly weak noncovalent interactions,
which give proteins stabilization energy, before secretion. One of these interaction is disulfide
bridges which is covalent bond between Cysteine residues. -S-H groups of two Cysteine
residues are firstly reduced, then they interact with each other in order to make oxidized -S-S-
bridge. In addition, it affects the e distance and angle between the C and S atoms of the
joined cysteine residues. Hence, unfolded state is destabilized via diminish in entrophy.
(Petsko & Ringe, 2004; Wedemeyer, Welker, Narayan, & Scheraga, 2000) In the case of weak
polar interactions (noncovalent interactions), they are bound up with electrostatic attraction
between positive and negative charges. There are also covalent interactions between folded
proteins. On the other hand, the effect of a this single interaction is not significant due to
small change in enthalpy, but folded protein structure contains hundreds to thousands of such
interactions that make significant contribution to enthalpy. The most essential ones are
hydrogen bonds and Van der Waals interactions. In the case of hydrogen bonds, (partially)
positively charged hydrogen atom bind covalently to donor atom which is more
electronegative (e.g. oxygen), then adjacent negatively charged acceptor atom is attracted. By
the help of these interactions, two non-hydrogen atoms close up and total radii forms
hydrogen bond. (Hubbard & Kamran Haider, 2010; Petsko & Ringe, 2004) In Van der Waals
interactions, the electron clouds of two atoms or atom groups weakly attract to each other, and
the force emerge from electron fluctuation around the nuclei form between atoms. The effect
of this interaction is higher in the proteins which contain methyl and/or methylene groups of
side chain (e.g. Valine and Leucine), and atoms must be close to each other in this interaction
because it is an extremely weak interaction. (Petsko & Ringe, 2004; Roth, Neal, & Lenhoff,
1996) In addition to these interactions, the hydrophobic effect is too important factor for
protein folding. Hydrophobic amino acids (e.g. Alanine and Valine) are clustered through the
core of protein, and charged and polar amino acids are move through the surface of the
protein when protein is folded. The decrease in amount of hydrophobic side chain which is
exposed to water is the main driving force for protein folding because protein structure
become more stable in folded state. (Tanford, 1978)

As primary, secondary, tertiary and quaternary, proteins have mainly four different structure.
Primary structure is the amino acid sequence aligned linearly in polypeptide chain. Secondary
structure forms repeating patterns named alfa helices, beta sheets and beta turns. Alfa helices
are coiled and right or left handed conformation, and N-H group of each residues (n) found
in backbone donate hydrogen bond to C=O of each residue (n+4). Beta sheets are formed
from the segments of prolonging peptide chain by formation of backbone hydrogen bonding
as parallel or antiparallel. Beta turns can be considered as tight turns which extend along
polypeptide chain in the reverse direction and it is stabilized via one or more backbone
hydrogen bond. In other words, secondary structure provides a great contribution to
stabilization of folded protein by hydrogen bonding that supply enthalpy of stabilization for
protein.(Petsko & Ringe, 2004) Tertiary structure is a 3D shape of the proteins which have
one or more protein domain and it is build with secondary structure proteins. Fibrous and
globular proteins are example of tertiary structure. Quaternary structure is the highest protein
organization that contains more than one polypeptide chain. These polypeptide chains which
forms quaternary structure interact with each other noncovalently. (Vaclavik & Christian,
2014)

SDS-PAGE is a technique that provides separation of protein with respect to its size and/or
shape, and provides analyzation of protein purity by denaturation of proteins. In other words,
a protein mixture can be separated into its components and they can be identified by
determination of their molecular weight. In addition, SDS-PAGE is used for detection of
protein modification, verification of protein concentration, detection of proteolysis,
identification of immunoprecipitated proteins and detection of protein modification. (Ahmed,
2005)

The basic mechanism of SDS-PAGE is based on denaturation of native protein with SDS and
other reagents (e.g. -mercaptoethanol which breaks the disulfide bonds), and it mask the
native protein surface charge, then gives negative charge to protein. So, charge/size ratio
become the same for all protein, and separation is performed only on the basis of size. This
technique consists of two gel systems: Stacking gel and Separating gel. In Stacking gel,
proteins are concentrated and aligned as a straight line to enter Separating gel at the same
time. In Separating gel as its name implies, proteins are separated with respect to its molecular
weight. The proteins which have high molecular weight move slower and drop back; low
molecular weight proteins move faster. After running process, the bands are compared with
the known sample bands (ladder) and their molecular weight is determined. (Ahmed, 2005)

SDS-PAGE is a common technique used for many research area. One of the research is about
seed protein in wheat which is Glutenin. To determine its allelic variation, effect on functional
properties and chromosomal location of genes, Low Molecular Weight (LMW) subunit must
be separated from the protein. Due to these reasons, LMW subunit is separated from Glutenin
and SDS-PAGE procedure is applied. Then, SDS-PAGE data is compared with SDS-PAGE
data for Gliadin which has similar extract-abilities and electrophoretic mobilities.(Singh,
Shepherd, & Cornish, 1991) Other research is about determination of raw meat of different
animal species from a mixture by comparison SDS-PAGE bands of special meat proteins.
(Ekii cii & Akyz, 2003) Another research is about pSUPER vector generated to control the
synthesis of siRNA in mammalian cells. By the help of this vector, stable loss of function
phenotypes forms by mediation of siRNA that leads to specific down regulation of gene
expression. These expression level is observed via SDS-PAGE. (Brummelkamp, 2002)

The alternative methods of SDS-PAGE for protein identification, determination and

separation are 2D-electrophoresis, immunoblotting, isoelectric focusing, Kjeldahl Method,
autoradiography, chromatography, mass spectrometry, Bioorthogonal Noncanonical Amino
Acid Tagging (BONCAT). (Dieterich, Link, Graumann, Tirrell, & Schuman, 2006; Li, 2007)
The alternative methods of SDS-PAGE for protein concentration determination are mass
spectrometry, Bradford Method, Biuret Method, Lowry Method, BCA, spectrophotometry.
(Thermo Scientific Pierce Protein Assay Technical Handbook, 2010). The alternative methods
of SDS-PAGE for protein modification determination are mass spectrometry, autoradiography,
antibody microarrays, Phos-tag Method, immunoblotting-based methods. (Johnson, 2012)

Egg albumin, BSA and and a-chemotrypsin contain disulfide bonds in their tertiary structure.
Egg albumin (ovalbumin) contains only one disulphide bond in 74-121 position. BSA
contains
17 disulphide bond in 77-86, 99-115, 114-125,147-192, 191-200, 223-269, 268-276, 288-302,
301-312, 339-384, 383-392, 315-361, 460-471, 484-500, 499-510, 537-582 and 581-590
position. a-chemotrypsin contains 5 disulphide bond in 1-122, 42-58, 136-201, 168-182, 191-
220 position. (Chymotrypsinogen A precursor - Bos taurus (Bovine), n.d.; ISHIMARU,
ITO, TANAKA, TANAKA, & MATSUDOMI, 2011; Patterson & Geller, 1977)

RESULTS
 Ladder 1.1 1.2 2.2 2.3 3.1 3.2 4.1 4.2
-chemotrypsin BSA Egg- albumin Total Protein

Fig. 1 SDS-PAGE data for -chemotrypsin, BSA, Egg- albumin and Total Protein (and protein
ladder)
https://www.thermofisher.com/order/catalog/product/26616

According to their molecular weight, the loading order is -chemotrypsin (left), BSA and
Egg- albumin (right). The molecular weight of -chemotrypsin is 25kDa, the molecular
weight of BSA is 66kDa and the molecular weight of Egg- albumin is 42.7kDa. Because high
molecular weight proteins are migrated slower, and low molecular weight proteins moves
faster, -chemotrypsin is the fastest and BSA is the slowest one.
DISCUSSION

In this experiment, the molecular weight of proteins was determined by SDS-PAGE. First of
all, SDS-PAGE system was prepared. The important point of this step is to properly place two
SDS-PAGE glass into the system to prevent leakage of gel mixtures. The second step is
separating gel (10%) preparation. All falcons must be properly labelled (e.g. separating gel
and stacking gel) before the experiment to prevent confusion (error). Acrylamide which is
monomer for polymerization reaction and bisacrylamide which is crosslinking agent for
polymerization were mixed with dH2O, then Tris-HCl buffer which provides ion to
environment and helps conduct electrical current was added. SDS was added to linearize
proteins (protein denaturation by disrupt hydrophobic interactions) and to give them negative
charge (In addition, -mercaptoethanol is used to break disulphide bridges). After APS which
is initiator of polymerization was added to mixture, TEMED which is catalyst of
polymerization (gives free radical for polymerization) was added immediately. Quick addition
of TEMED is too important because polymerization takes place rapidly, and polymerization
cannot be achieved properly without TEMED. To get homogeneous mixture, tube was
inverted gently. Then, separating gel mixture was poured between two SDS-PAGE glasses,
and isopropanol was added onto the gel to prevent oxygen penetration because oxygen inhibit
polymerization. In addition, it is important to leave some gel mixture in falcon to observe
polymerization process. Separating gel is used for separation of proteins with respect to their
size. The third step is stacking gel (4%) preparation. The same procedure was applied for
stacking gel with different amount. However, isopropanol top of the separating gel must be
removed before adding APS of stacking gel to provide continuous gel system. Then, APS and
TEMED can be added to mixture. Before pouring stacking gel, it should be waited for
completion of separating gel polymerization. Comb was inserted to system to form well after
pouring stacking gel. The same critical points are valid for stacking gel. Stacking gel is used
to align proteins as straight line, and it allows protein to enter separating gel at the same time.
The third step is sample preparation. After polymerization of stacking gel, proteins were
mixed with loading dye and mixture was waited in hot water bath to improve denaturation,
then spin down process was done to collect all sample in one place. Loading dye includes
glycine (to make proteins heavier and to help them to sink in gel), protein and
bromophenolblue (tracking dye to prevent fall protein down gel system). Sample and
molecular weight marker application to wells is to important, and the person who apply
sample must be careful to prevent gel cracking and diffusion of sample to the gel. 20A was
firstly applied to system for stacking gel running, then 40A was applied for separating gel
running. Also, pH of gels are important for running rate of samples and loading dye. In
stacking phase, glycine moves slower and BPB moves faster. Both glycine and BPB squeeze
proteins and carry them. Whereas, glycine moves faster in separating gel and they are
separated each other. The gel was run until tracking dye was migrated to within 2-5mm of
bottom of separating gel to prevent fall down sample. For staining process, gel was put into
staining solution which contains Coomassie blue (stain peptide bonds), methanol and ethanol
(help fixing dye). After the experiment, molecular weight of proteins was determined by
comparison with molecular weight marker and protein bands.(A Guide To Polyacrrylamide
Gel Electrophoresis And Detection, n.d.)

Moreover, two different dye was used for the same proteins. When looking at the bands, there
is a difference between two bands for the same protein in terms of band distance travelled by
the same protein. The reason of this difference is the lack of -mercaptoethanol in one of the
dyes. -mercaptoethanol is agent that causes disruption of disulphide bridges in protein. When
looking at the disulphide bridge number of the proteins mentioned in introduction, BSA has
highest bridge number, so absence of -mercaptoethanol affects BSA more. On the other
hand, Egg albumin has only one bridge. Because of this, it is not affected as much as BSA
from absence of -mercaptoethanol (because protein folding is not too much due to small
number of disulphide bridges) as it can be easily seen in bands. Also, the difference in folded
and unfolded proteins can be seen by comparison with total protein bands.

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