Fish Muscle Protein Extraction SDS-PAGE

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 45Kc.
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
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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.