Fish Muscle Protein Extraction SDS-PAGE

This experiment focuses on the extraction of fish muscle protein using SDS-PAGE technique. It explains the process of denaturing proteins with SDS to separate them based on size. The results show the relative positions of protein bands on the gel and how to identify specific proteins based on their molecular weight. The experiment provides insights into the principles of protein separation and analysis.

EXPERIMENT (1) - FISH MUSCLE PROTEIN EXTRACTION SDS-PAGE
A PAGE (polyacrylamide gel electrophoresis), is an analytical method used to separate
components of a protein mixture based on their size. The technique is based upon the
principle that a charged molecule will migrate in an electric field towards an electrode with
opposite sign. The general electrophoresis techniques cannot be used to determine the
molecular weight of biological molecules because the mobility of substance in the gel
depends on both charge and size. To overcome this, the biological samples needs to be
treated so that they acquire uniform charge, then the electrophoretic mobility depends
primarily on size. For this different protein molecules with different shapes and size, needs to
be denatured (done with the aid of SDS) so that the proteins lost their secondary, tertiary or
quaternary structure.
The proteins being covered by SDS are negatively charged and when loaded onto a gel and
placed in an electric field, it will migrate towards anode (positively charged electrode) are
separated by a molecular sieving effect based on size. After the visualization by staining
(protein specific) technique, the size of a protein can be calculated by comparing its
migration distance with that of a known molecular weight ladder (marker).
RESULTS –
1.The relative positions of the bands on the gels indicate the relative molecular weight of the
protein.High molecular weight proteins are on the above and low molecular weight proteins
are located below relative to the ladder.
2.As from the image available we can explain the differences in size and intensity of bands of
the protein by comparing them with a pre-stained protein ladder.The big molecular weight
proteins remain on top and the low molecular weight proteins they come to the bottom.
3.Specific proteins can be identified from the image by knowing their molecular weight , for
example the fish muscle protein of Alaskan Pollock is expected to give a band around at 25.5
Kda so by knowing their molecular weights we can easily identify the protein to be isolated.
EXPERIMENT (2) : LECTIN EXTRACTION -SPECTROPHOTOMETRIC
ANALYSIS OF PROTEINS
Spectrophotometry is one of the most widely used analytical procedures in biochemistry. It is
commonly used to estimate the level of an analyte in a solution and is ideal for simple routine
determination of small quantities of materials. This method is based on two laws of light
absorption by solutions, namely LAMBERT'S LAW AND BEER'S LAW.
LAMBERT'S LAW states that "the proportion of light absorbed by a medium is independent
of the intensity of incident light” while BEER'S LAW maintains, “the absorbance of light is
directly proportional to the concentration of the absorbing medium and the thickness or path
length of the medium".
LAMBERT'S LAW is expressed as:
TRANSMITTANCE (T) = INTENSITY OF TRANSMITTED LIGHT(I) / INTENSITY OF
INCIDENT LIGHT (I•)
BEER'S LAW is expressed as :

ABSORBANCE (A) = EXTINCTION COEFFICIENT OR MOLAR ABSORPTIVITY(e) ×
CONCENTRATION (C) × PATH LENGTH (L)
Since proteins absorb light at a specific wavelength, a spectrophotometer can be used to
directly measure the concentration of a purified protein in solution. It is important to note that
direct UV measurement at 280nm yields highly reproducible measurements since no reagents
are added to the protein solution and the protein of interest was not modified or inactivated
during the process. It also produces quick results since the samples does not need to be
incubated in order to complete the process.
CALCULATING PROTEIN CONCENTRATIONS
To commute for the concentration of a purified protein in a solution, the following formula
can be used:
Concentration (mg/ml)= A_280 × conversion factor.
RESULT
1.The spectrophotometric values 260 and 280 gives the purity of the isolated protein
samples.So for pure protein samples A260/280 is 0.57.
2.The given images shows the gel images of two protein samples taken at 30 and 45ͦc.
3.In the experiment presence of lectins was confirmed by the assay.The isolated proteins
were then subjected to further purification methods.
EXPERIMENT [3] - YEAST SHOCK HEAT PROTEIN BY BRADFORD ASAAY
AND WESTERN BLOT
Western blotting is a widely used technique for the detection and analysis of proteins based
on their ability to bind to specific antibodies. Western blotting is accomplished rapidly using
simple equipment and inexpensive reagents. It is a commonly used laboratory technique. The
specificity of antigen antibody interaction enables to a target protein to be identified in the
midst of a complex protein structure.
It is an analytical method where in protein sample is electrophoresis on an SDS- PAGE and
electrophoresis transferred onto PVDF membrane or nitrocellulose membrane .The
transferred protein is detected using specific primary and secondary enzyme labeled antibody.
Antibodies bind to specific sequences of amino acids, known as the epitope, because amino
acids sequences are different form protein-to-protein, antibodies can recognize specific
proteins among a group of many. Therefore, a single protein can be identified in a cell lysate
that contains thousands of different proteins and its abundance quantified through western
blot analysis. First, proteins are proteins are separated based on their size. Second, antibodies
are used to detect the protein of interest. Finally, a substrate that reacts with an enzyme is
used to view the antibody/protein complex.
BRADFORD assay is a quick spectroscopic analytical procedure that is used to measure the
concentration of protein in samples.The r2 square value in the graph is coming to be around
0.8 which shows that curve is good to determine protein concentration.

0 500 1000 1500 2000 2500
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8 R² = 0.816936668971659
BRADFORD ASSAY
Image of stained SDS-PAGE gel
No difference was observed in the overall loading of samples.
No change in intensity was observed in both the samples.
EXPERIMENT (4) MICHAELIS -MENTEN
The MICHAELIS -MENTEN model is one of the simplest and best-known approaches to
enzyme kinetics. It takes the form of an equation relating reaction velocity to substrate
concentration for a system where a substrate S binds reversibly to an enzyme E to form an
enzyme substrate complex ES , which then reacts irreversibly to generate a product P and to
regenerate the free enzyme E . This system can be represented schematically as follows:
E + S= ES= E + P
The Michaelis Menten equation for this system is
V= Vmax [S]/Km + [S]
Here Vmax represents the maximum velocity achieved by the system, at maximum substrate
concentration .Kmax ( the michaelis constant, sometimes represented as Ks instead) is the
substrate concentration at which the reaction velocity is 50% of the Vmax [S] is the
concentration of the substrate S.

EXPERIMENT : DNA SECTION LAB REPORT
EXPERIMENT 1: ISOLATION OF DNA
High molecular weight DNA is readily degraded by shear forces and so extreme care should
be taken during its isolation. Mechanical stress including rapid stirring and extreme physical
and chemical conditions such as vortexing must be avoided as they would break the high
molecular weight DNA into much shorter fragments. Therefore DNA is isolated from the
cells by the gentlest possible method of cell rupture in the presence of EDTA to chelate Mg2+
which is essential for DNase activity.
The protein is denatured by treatment with phenol-chloroform and the DNA is precipitated
with ethanol. The product is then dissolved in a buffer and stored at 40C over a drop of
chloroform.
Nucleic acids absorb strongly in the UV region of the electromagnetic spectrum due to
conjugated double bond systems i.e. the constituent purines and pyrimidines. For estimation
of DNA the absorbance should be measured at wavelengths of 260 nm and 280 nm. A pure
preparation of DNA has an A 260/280 ratio of 1.8.
Cloning, Expression and Purification of a Gene Product in E.coli:
In week 2 after isolation of DNA, the isolated DNA was used for further process of cloning
and transformation.One of the fundamental steps in understanding the role of various genes
of either prokaryotes or eukaryotes involves the cloning, expression and purification of those
genes product. Cloning and expression of these genes in E. coli has proved to be very useful
for a number of reasons. The physiology, genetics, vector systems used for propagation in E.
coli is well known. It is cheap and easy to grow with a generation time of only 20 min. Its
culture media is well defined and large amounts can be grown in fermenters. However there
are certain problems also faced with its use as there is no splicing of introns/exons, only
cDNAs can be used for the expression of eukaryotic genes and foreign promoters do not
work in E. coli. So promoter from E. coli gene (-35, -10 sequences) is needed along with the
bacterial ribosome binding site. Various promoters that have been incorporated into a number
of expression plasmids include:
1. trp promoter: from tryptophan biosynthesis operon, is controlled by tryptophan in
media, acts as a stronger promoter than lac & has better ribosome binding site.

2. tac or trc promoters (1 bp difference) : have -10 sequence from lac promoter, are
controlled by IPTG, have -35 sequence from trp promoter and show no catabolite
repression.
3. phage promoter: e.g λ pL promoter which is under control of cI repressor and uses
cIts857 mutant which is a temperature-sensitive mutant and induces the expression at
40°C.
4. T7 promoter: from T7 phage which is only expressed by T7 RNA polymerase in
vitro or in vivo and requires cloned T7 RNA polymerase gene to regulate expression
in vivo. It is also possible to regulate T7 RNA polymerase expression using lac, trp, or
λ pL promoter thus allowing only cloned gene expressed at high level. The advantage
of this system is that regular E. coli genes are not recognized by phage RNA
polymerase.
To overcome this problem cloning is done in protease deficient that is lon mutant strains. It is
also seen that over-expression of a protein leads to the formation of inclusion bodies which
are insoluble aggregates of protein. These are easy to purify by lysing cells and pelleting
aggregates by centrifugation followed by denaturation and finally refolding into active form.
However in many cases the refolding may not necessarily yield active form of the protein.
Addition of “tags” (protein sequences that bind specific ligands) can be done for purification
of protein from either the soluble (properly folded) or insoluble fraction. These tags may be
removed by cleavage with certain enzymes or chemicals.
Another problem with cloning in E. coli is that of codon usage. It is known that different
organisms use codons with different frequencies which depend on GC content, methylation
sites as the tRNA frequencies are optimized for codon use. Hence translation of cDNA in E.
coli will stall at these codons. But these problems are overcome by changing to common E.
coli codons by site directed mutagenesis or by synthesizing artificial gene.
To amplify the gene of interest by PCR:
The polymerase chain reaction (PCR) is a powerful molecular biological method with
widespread applications in genetics and disease diagnosis. It is an in vitro method of nucleic
acid synthesis by which a particular segment of DNA can be specifically replicated. It
involves the use of two oligonucleotide primers, which typically have different sequences and
are complimentary to the flanking region of the sequence to be amplified. The primers anneal

to the opposite strands of the template DNA. The template DNA is first denatured by heating
in the presence of a large molecular excess of each of the two oligonucleotides and 10 mM
dNTPs. The reaction is then cooled to a low temperature that allows the oligonucleotide
primers to anneal to that target sequence, after which the annealed primers are extended with
DNA polymerase. Since the extension products themselves are also complementary to and
capable of binding primers, a successive cycle of denaturation, annealing and DNA synthesis
is repeated many times. Because the product of one round of amplification serves as
templates for the next, each successive cycle essentially doubles the amount of the desired
DNA product.
TAILING AND LIGATION OF THE PCR PRODUCT
Since thermostable DNA polymerases with proofreading activity, like Pfx polymerase,
produce blunt ended PCR products and for cloning in the pGEM®T vector, an ‘A’ overhang is
required, hence the purified blunt ended PCR product is modified. A 10 l reaction will be
put up using of Taq DNA polymerase; 1X PCR buffer; 1.5 mM MgCl2; 0.2 mM dATPs and
purified insert. The reaction mixture will be incubated at 72oC for 30 minutes and the
required amount of insert will be directly used for ligation with linearized pGEM®T vector.
The pGEM®-T Vector Systems are convenient systems for the cloning of PCR products. The
vectors are prepared by cutting the pGEM®-5Zf (+) and pGEM®-T Easy Vectors,
respectively, with EcoRV and adding a 3´terminal thymidine to both ends. These single 3´-T
overhangs at the insertion site greatly improve the efficiency of ligation of a PCR product
into the plasmids by preventing recircularization of the vector and providing a compatible
overhang for PCR products generated by certain thermostable polymerases. Taq Polymerase
add a single deoxyadenosine, in a template-independent fashion, to the 3´-ends of the
amplified fragments.
Transformation of E. coli
Introduction of an exogenous DNA into a suitable bacterial host is termed "Transformation".
It is a modified procedure of that followed by Mendel and Higa. In this procedure, E. coli
cells and DNA interact in an atmosphere of divalent cations eg. Ca2+, CaCl2 enhances the

competence of the cells to take up DNA resulting in yields of transformants as high a 10 6 per
μg of DNA.
Several conditions such as heat shock treatment of the cell / DNA mixture for a brief period,
the inclusion of the monovalent cations in the transformation buffer the addition of
hexaaminecobalt chloride, the treatment of cells with the solvent and some sulfhydryl
reagents and growth in a medium containing higher levels of magnesium (10-20mM) have
considerably improved the efficiency of transformation provided the reagents employed are
pure. Transformation is carried out in two steps:
a. Preparation of competent cells and
b. Transformation by plasmid DNA or the ligated DNA mix
Preparation of expression vector DNA by alkaline lysis with SDS: Mini preparation
Plasmids are double stranded self replicating circular DNA molecules. Many methods have
been developed to isolate and purify plasmid DNA from bacteria. These methods invariably
involve three steps.
1. Growth of the bacterial culture
2. Harvesting and lysis of the bacteria
3. Purification of plasmid DNA.
Plasmids are almost always purified from O/N cultures grown in liquid media containing
appropriate antibiotic. Bacteria are recovered by centrifugation and suspended in an isosmotic
solution of sucrose and then treated with lysozyme and EDTA to break down the cell wall
and outer membrane. The resulting spheroplasts are lysed by treatment with detergents and
alkali. These treatments disrupt base pairing and cause the linear chromosomal DNA of the
host to denature. However, the strands of covalently closed circular (CCC) plasmid DNA are
unable to separate from one another. When conditions are returned to normal the strands of
plasmid DNA rapidly fall into perfect register.
Cutting and joining of DNA
Duplex DNA can be cut at sequence specific sites by restriction endonucleases. There are
three types of restriction endonucleases namely type I, type II, and type III. Out of these, type

II enzyme which recognize specific sequences are widely used in cutting the DNA for gene
cloning. Over 600 restriction enzymes from different sources are known today.
DNA fragments from several different sources can be ligated to produce recombinant
molecules. Ligation is the process by which DNA molecules are joined in the presence of the
enzyme ligase. In the recombinant DNA experiments two major types of ligases are
employed. These are E.coli DNA ligase and phage T4 encoded ligase. Both these ligases seal
single stranded nicks between adjacent molecules in the duplex state by forming
phosphodiester bonds. ATP is essential for the activity of T4 enzyme which can ligate both
cohesive ends and blunt ended fragments whereas the E.coli enzyme acts only in the presence
of annealed cohesive ends and requires NAD.
The ease with which each DNA fragment is cloned into plasmid vector depends on several
factors. Cloning is considerably more successful when only one DNA fragment is to be
ligated into a vector. Efficient cloning occurs when the vector and insert DNA contains
complementary overhanging ends and less efficient when both the fragments are blunt. The
highest cloning efficiency is achieved with DNA cleaved by two different restriction enzymes
that produce noncomplementary ends. Ligating templates prepared in this manner is referred
to as directional cloning as insert DNA can be ligated into vector in only one orientation.
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