PROTEIN EXTRACTION AND QUANTIFICATION
Juan Victor Gamo, Rafaella Giana Javier, Joseph Raymund Sanchez, Tanya Cecille Valdez
CH152-B
Abstract
Proteins.....
INTRODUCTION
Proteins are long chains, or polymers, of
amino acids linked by peptide bonds. The folding and
twisting of these chains form their conformational
structures which give proteins their distinctive
properties. They are considered the workhorse of the
cell because of their varying functions. (Pratt, 2014).
These functions include providing structural stability,
acting as motors for movement and participating in
the expression of genetic information, among others.
Figure 1:
Figure 2:
METHODOLOGY
A. Compounds Used
- DNA extracted from raw chicken liver
- distilled water
- deionized water
- 0.5M EDTA (C10H16N2O8+) solution
- 0.25M sodium hydroxide (NaOH) solution
- 0.25M hydrochloric acid (HCl) solution
- SYBR stain
- bromophenol blue
- sucrose
- Tris (C4H11O3N)
- Glacial acetic acid (CH3COOH)
- powdered agarose
- human saliva
B. Procedure
Preparation of Buffers
To prepare 6X gel loading buffer
(GLB), 0.100g of bromophenol blue and 1.6g sucrose
were added together and brought to 4mL. This was
then pipetted into each of the microfuge tubes. The
1X Tris-Acetate-EDTA (TAE) running buffer was
prepared using 4.84g of Tris, 1.14g of glacial acetic
acid, 2mL of 0.5M (at ph 8.0 which was prepared
beforehand) and was brought to 1L.
DNA Degradation
Due to lack of proper equipment and time
constraints, spectrophotometric analysis could no
longer be done in the laboratory. Instead, the DNA
stored in the microfuge tubes were subjected to
different conditions to examine DNA degradation,
namely; control, boiling, mouth nucleases, acid
treatment, and alkali treatment. For the control
microfuge tube, no treatment has been done. The
microfuge tube assigned for boiling was suspended in
a boiling water bath for approximately five minutes
and was allowed to sit at room temperature for ten
minutes. Two microliters of saliva (from one of the
experimenters) was added to the microfuge tube
assigned to Mouth Nucleases and was mixed together
with the DNA and incubated at 37 degrees for 20
minutes. For the Acid treatment microfuge tube, 1 uL
of 0.25N HCl was added and mixed together. This was
then incubated and 37 degrees for about five minutes.
The same process was done with the Alkali Treatment
microfuge tube but instead of HCl, 1uL of 0.25N NaOH
was added. Each microfuge tube was labeled
accordingly for easy identification.
Agarose Gel Electrophoresis
In a 250mL Erlenmeyer flask, powdered
agarose was added to the 1X TAE running buffer to
make 100 mL of 0.6% solution. SYBR stain was also
added to the solution. The slurry was heated until the
agarose dissolved, creating a clear solution. This was
then cooled to 60 degrees while carefully swirling the
contents to ensure even temperature and
concentration. The combs were positioned on the
plates before pouring the warm agarose solution to
the molds. The gel width was about 3-5 mm thick.
After the gel had solidified, the comb and the casting
dams were very carefully removed, ensuring the wells
were still intact. Electrophoresis buffer was added to
cover the entire gel to a depth of about 2 mm.
Afterwards, the DNA samples in the
microfuge tubes were mixed with 5µL of GLB and
vigorously swirled (due to the lack of a mini
centrifuge). With a yellow-tip micropipette, the
mixtures were loaded into the wells (about 20 µL per
well). The gel was run at a constant of 100V for 30
minutes.
RESULTS, DISCUSSION, and CONCLUSION
After each DNA degradation procedure, the
samples were run through AGE. At the end of the
agarose gel electrophoresis procedure, the following
band patterns were observed in the gel. The following
figure shows the DNA samples before and after
running the electrophoresis machine:
(A) (B)
Figure 3. The DNA samples before and after AGE. Figure
3A shows the samples after each treatment in 5 separate
wells (From left to right the treatments are: Control,
Boiling, Mouth Nucleases, Acid, and Base). Figure 3B
shows the resulting band patterns after 30 mins.
From the results indicated in Figure 2, it can
be seen that no noticeable DNA bands were formed
after performing AGE. What could be concluded from
this result was that not enough DNA was present in
the samples to result in noticeable band patterns for
the different treatments. As a result, no final
conclusion could be made regarding the effects of the
different treatments on the integrity of the DNA
structure. However, based on available data from
existing literature, it was expected that during the
heat treatment and mouth nuclease treatment of
DNA would have resulted in shorter DNA fragments as
compared to the control treatment. This is because in
response to heat, the H-bonds holding DNA together
would be overcome and the DNA may be broken
down into smaller fragments due to these bonds
being broken (Klug, Cummings, Spencer, & Palladino,
2012). In response to the mouth nucleases, the DNA
would also be expected to produce bands closer to
the cathode since the nucleases would also cut the
DNA into shorter sequences. The sonication, acid,
base, and freezing treatments would have also been
expected to result in shorter DNA fragments that
would have produced bands closer to the cathode as
compared to the control. For sonication, the H bonds
in the DNA would be expected to be disrupted leading
to DNA fragmentation, while for the freezing
treatment, the physical stress of ice crystals on the
DNA strands as well as possible pH changes due to the
widely varying temperatures the DNA would be
exposed to would also lead to DNA fragmentation
(Elsner & Lindblad, 1989). The large change in pH to
the environment of the DNA in the acid and base
treatments would have also led to hydrolysis of the
phosphodiester bonds linking the different
nucleotides together and so DNA fragmentation was
also expected to occur. For the UV treatment, longer
DNA strands were expected to form and as a result,
the UV-treated DNA would be expected to produce
bands located higher than the control treatment. This
is due to the UV light causing the formation of
thymine dimers in the DNA thus leading to lesions and
bulging in the overall structure of the DNA (Klug,
Cummings, Spencer, & Palladino, 2012).
Other possible sources of error during the
execution of the experiment was that the analysis of
the DNA purity via spectroscopy was no longer
performed. This made it impossible to accurately
judge beforehand whether or not the DNA samples
were heavily contaminated with other proteins or not
prior to electrophoresis. It was important to maintain
proper sterile techniques all throughout the
experiment while handling biological samples so that
external contaminants such as oil and skin and dust
from the experimenters' hands or from the air would
not contaminate the sample by adding other proteins
or oils that may be mixed in together with the DNA
However, since a quantitative means of confirming
the purity of the DNA samples was no longer
performed, the purity could not be absolutely
guaranteed when AGE was being carried out, and so
contamination may have likely played a part in
causing no bands to form.
Another possible source of error could have
occurred during the staining procedure. Instead of
using the SYBR stain, it is possible that other
alternative may have produced better band patterns
such as using the Fastblast DNA stain to coat the gel
after the electrophoresis or also the use of ethidium
bromide (EtBr) to produce bands that could be viewed
under a UV transilluminator. The advantage of using
the EtBr stain would be that it would be able to better
stain the DNA molecules and it would give clearer
images of the bands since it would still be viewed
under UV. The disadvantage of EtBr is that is an
intercalating agent and is carcinogenic when exposed
to bare skin. Using the Fastblast staining method
would have been much safer as compared to EtBr but
it would taken a much longer time to complete and
the there may have been risk of overstaining the gel.
It is likely however the most likely reason for
the absence of noticeable DNA bands patterns after
the electrophoresis was that there was simply Edition. Glenview, IL: Pearson Benjamin
insufficient amounts of DNA contained within the Cummings.
samples. It would be recommended that PCR would
have been performed prior to AGE in order to amplify University of Alberta. (n.d.). An Introduction to DNA:
the amount of DNA present in each of the samples Spectrophotometry & Degradation. University
before running through the gel to better ensure that of Alberta Biological Sciences. Retrieved
more noticeable band patterns would be formed. October 1, 2016, from
Since there was very few DNA present in the samples, http://www.ableweb.org/volumes/vol-22/5-
after the electrophoresis procedure, it was likely that clark.pdf
all of the DNA had been washed off of the gel and so
no noticeable bands were formed. Had PCR been
performed, a large amount of the liver DNA would
have been produced prior to the DNA degradation
treatments and since more DNA would have been
present prior to electrophoresis, the likelihood of all
of the DNA being washed off the gel would be
reduced and so more would be left on the gel at the
position expected corresponding to the length of the
DNA strand (Klug, Cummings, Spencer, & Palladino,
2012). Apart from DNA amplification to ensure a
sufficient amount of DNA is present for AGE, another
importance of PCR is that when a particular gene of
interest has been isolated or obtained, PCR is a useful
method to produce many copies of this gene of DNA
sequence of interest so that re-extraction from the
source organism would no longer have to be
performed every single time a particular gene or DNA
sequence is to be studied.

In conclusion, since no noticeable bands were

formed as a result of insufficient DNA being present in
the samples, no definite conclusion can be made
regarding the experimental effects of the various
degradation treatment procedures on the integrity of
the DNA sample from a raw chicken liver. While some
of the expected effects were indicated by existing
literature the effects of the specific treatments used
in the experiment could not be determined. It is
recommended that a proper DNA amplification
procedure such as PCR be performed prior to
electrophoresis in order to better guarantee that
noticeable DNA bands would form on the agarose gel.
It is also recommended that an assessment of the
purity of the DNA sample be performed immediately
after extraction so that it could be determined right
away whether or not DNA would have to be extracted
again from the sample due to possible
contaminations.

REFERENCES

Elsner, H., & Lindblad, E. (1989). Ultrasonic

degradation of DNA. DNA, 8(10), 697-701. doi:
10.1089/dna.1989.8.697.

Klug, W.S., Cummings M.R., Spencer, C.A., & Palladino

M.A. (2012). Concepts of Genetics 10th