From what we call the building
blocks of life or protein, are smaller
units called amino acids. Out of twenty
existing amino acids in this world, one
characteristic is shared among all these
amino acid: the presence of an amino
and carboxyl group as well as their R-
group side chain, which are primarily
used for their classification
additionally, for the characterization of
both free amino acids and protein since
amino acids have a wide array of
chemically reactive groups (Bathan, et al.
2017).
To identify their presence in the
biological media, qualitative
reaction tests were designed.
example of this is the Ninhydrin Test,
which tests for the amino acids with an
alpha-amino group, and gives a blue to
blue-violet coloration to indicate
positive result. Another example is the
Xanthoproteic Test for the aromatic
acids, namely Tryptophan
Phenylalanine (F) and Tyrosine (Y).
Positive result gives off a yellow solution.
Another test, Millon’s Test, is for the
detection of amino acids with phenolic
ring (Tyrosine) which shows an old rose
to fleshy red coloration for positive result.
For the detection of amino acids with an
indole group, Hopkins-Cole Test is the
one to be used. This gives off a pink to
violet interface if the result is positive.
Sakaguchi’s Test, on the other hand, is
for the detection of amino acids with un-
or monosubstituted guanidine (Arginine)
and which consequently shows a red to red-
orange color for a positive result. Fohl’s
Test, specifically for sulfur-containing
amino acids (Cysteine and Methionine),
gives off a brown to black precipitate for
positive result indication. For the
detection of Cysteine alone,
Nitroprusside Test is the one to be used.
colour
Positive result in this test can be inferred
One
if red coloration has occurred. Test for
amides, which is designed to observe
primary, secondary and tertiary amides
and nitriles, will turn a red litmus paper
a
to blue if positive. Another test is the
Pauly’s Test for the detection of Tyrosine
and Histidine. If results are positive, red
(W),
coloration will be eventually seen. Lastly,
the Biuret Test, for the observance of
intact proteins, protein hydrolysates and
peptide bonds, will give off a pink-violet
coloration if results happen to be positive
(Villalobos & De Ocampo, 2019.)
 Without proteins, there would be
no synthesis nor repair of DNA which
eventually lead to our own
mutations and problems, such as cancer
(Ede, 2013.) Some proteins in our body
generate hormones, including the FSH,
insulin, cholecystokinin, and many more
(Cook, 2019.) These are some of the
reasons why proteins are indeed vital for
our survival. Without proteins, our body
would have to disintegrate our own
muscle fibers in order to survive. This will
eventually lead to muscle atrophy or
muscle wasting. Protein-rich
includes milk, egg, soy and meat.
Casein, a globular milk protein
found in milk, is helpful in recovery and
reducing muscle breakdown (Mawer,
2016.) Other globular milk proteins are
lactalbumins and lactoglobulins.
biological analysis, the isolation of Casein
is performed through the adjustment of
the pH level to 4.6, which is the
isoelectric point of Casein, meaning,
there is no net charge in this pH value
since all the positive and negative
charges in casein are balanced (Acosta,
et al., 2015.)
Found in meat on the other hand,
are globular muscle proteins called
DNA myoglobin. Myoglobin is responsible for
storing oxygen for future use. It is an iron
and oxygen binding protein found in the
tissue of vertebrate muscles. To isolate
myoglobin, salt-induced precipitation
could be used, wherein ammonium
sulfate is used on the buffered muscle
extract. Salt-induced preparation, also
known as “salting-out or purification,
depends on the concept of protein
solubility that utilizes the decreased
food solubility of most proteins in high salt
concentrations. Another example of
globular muscle protein is hemoglobin,
which is found in the cytosol of red blood
cells in the bloodstream (Villalobos & De
Ocampo, 2019), is also responsible for
the transportation of the oxygen stored
For
and for the red color of tissue. However,
the difference between these two is that
hemoglobin can bind to carbon dioxide,
while myoglobin cannot.
Protein isolation in general is
performed to discover and know the
amino acids, beneficial or not, present in
our food. Other than isoelectric pH
adjustment (casein isolation from
skimmed milk), there are other ways for
protein isolation that can be performed
as well, such as salting-out (myoglobin
isolation from beef) , heat denaturation
(albumin isolation from skimmed milk),
difference in solubility in water (gluten
isolation from wheat flour),
chromatography, ultra centrifugation,
and many more.

The Thin Layer Chromatography

(TLC) is a procedure used for the
determination of how many compounds
are there in a particular mixture, their
polarity and purity. It can also be used,
in this experiment, for isolation and
identification of the components present
in the acid, alkaline, and enzymatic
hydrolyzed protein. Separation of amino
acids in TLC depends on the concept of
differential affinity of the solute including
the amino acid it had dissolved, between
the mobile and stationary phase.