EXPERIMENT NO.

1 & 2
Total Protein Isolation and Quantification
2015-46211, 2015-04570, 2015-11129
Biochem 124.1 HEG
23 September 2019

ABSTRACT
Analysis of proteins involves protein isolation, purification and characterization. Thus, in the experiment, protein
from different growing stages of mongo beans were primarily extracted and then quantified. The isolation of each
sample was done by initially freezing the protein samples with dry ice. This was followed by grinding,
homogenization using TRIZOL® reagent, fractionation of cell components using chloroform, and finally protein
precipitation using ethanol. The protein isolated was dissolved in 1% SDS for the succeeding quantification. Using
Bradford assay, a standard curve was generated with an R​2 of 0.7643. Sample quantification then revealed the
concentrations of seed, two-day old sprout, and four-day old sprout at -0.0107, -4.124 x10​-4​, and -5.051 x10​-3
mg/mL, respectively. These negative values are due to a lower actual protein concentration than the standards
used for the linear regression. Possible sources of error in the isolation procedure can be due to the
contamination of protein pellet or the incomplete solubilization of the protein precipitate. With this, it is
recommended to generate a standard curve with better linearity. In addition, polyvinylpolypyrrolidone (PVPP) is
also recommended to be added to TRIZOL® to reduce protein degradation by phenolics.

Keywords: protein isolation, mongo beans, TRIZOL®, protein quantification, Bradford reagent

1 INTRODUCTION only has a single reactive, rapid reaction, high

stability of the protein-dye complex, high
All biochemical investigations of biological samples reproducibility, and minimal interferences. Therefore,
require the isolation, purification, and in this experiment, Bradford assay was done to
characterization of a protein (Boyer, 2000). This is quantify the proteins extracted from different growth
followed by multiple downstream assays that would stages of mongo beans.
generate data depending on the objectives of said
assays. Thus, it is important to first isolate the target Overall, proteins from different stages of mongo
protein before all other analysis can be done to it. beans were isolated using TRIZOL® reagent. The
mode of action of each of the reagents used in
Essentially, all procedures in protein extraction protein extraction were identified. The isolated
follow these steps: selection and preparation of the proteins were then quantified via Bradford assay.
protein source, homogenization, fractionation of cell Furthermore, the principles behind dye-based
components, and protein precipitation (Boyer, 2000). absorbance measurements and colored complex
In this experiment, the methodology by the group of measurements for protein quantitation were also
Chomczynski (1993) was followed wherein discussed.
TRIZOL® have a key role in the overall extraction of
protein. TRIZOL® is a solution of phenol and 2 METHODOLOGY
guanidine isothiocyanate capable of isolating three
biomolecules simultaneously: DNA, RNA, and The isolation procedure was subdivided into seven
protein (Likhite & Warawdekar, 2011). steps: preparation of protein samples,
homogenization, phase separation, DNA
Once protein is isolated from its source, precipitation, protein precipitation, protein wash, and
quantification can be done. Some of the most protein pellet redissolution. Protein quantification
common procedures are the assays of Biuret, followed after isolation. It was subdivided into two:
Bradford, Kjendahl, Lowry, Smith, and construction of standard calibration curve and real
Warburg-Christian (Bonjoch & Tamayo, 2001). sample quantitation.
However, the spectrophotometric Bradford assay is
recommended by most researchers because of its
advantages. Five of which were enumerated by
Bonjoch and Tamayo (2001); that is, Bradford assay

Biochemistry 124.1 Total Protein Isolation and Quantification ​ age 1 of 7

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2.1 Preparation of Protein Samples the initial homogenization. The samples were stored
for 10 minutes at 15-30°C. The protein was then
The mongo bean samples were prepared in different precipitated at 12000×g for 10 minutes at 2-8°C.
stages: seeds, sprouts (1-2 days), and adults (3-4
days). The sprout and adults were prepared by 2.6 Protein Wash
planting the seeds on cotton placed in a sturdy
container. They were then watered at least once a The protein pellet was stored in the wash solution for
day with 5 mL of the watering solution. 20 minutes at 15-30°C. Then, it was centrifuged at
7500×g for 5 minutes at 2-8°C. The protein pellet
The fresh samples were placed in a stainless steel was washed three times in a solution containing 0.3
mortar and pestle along with dry ice and were then M guanidine hydrochloride in 95% ethanol. During
ground. Approximately 0.1 g of the sample as each wash cycle, 2 mL of wash solution was added
weighed in a pre-sterilized MCT. The samples were per 1 mL TRIZOL® Reagent used for the initial
stored at -20°C. homogenization. After the final wash, the protein
pellet was vortexed in 2 mL of ethanol. It was then
2.2 Homogenization stored in ethanol for 20 minutes at 15-30°C and
centrifuged at 7500×g for 5 minutes at 2-8°C.
The ground tissue samples were homogenized in 1
mL of TRIZOL® Reagent per 50-100 mg of tissue, 2.7 Protein Pellet Redissolution
making sure that the sample volume did not exceed
10% of the volume of the reagent. The insoluble The protein pellet was vacuum-dried for 5-10
material was removed from the homogenate by minutes. It was then dissolved in 1% SDS by
centrifugation (12000×g for 10 minutes at 2 to 8°C). pipetting. Complete dissolution of the protein pellet
the cleared homogenate solution was transferred to required incubation of the sample at 50°C followed
a fresh tube. by sonication for 2 hours. Any remaining insoluble
material was sedimented by centrifugation at
2.3 Phase Separation 10000×g for 10 minutes at 2-8°C. The supernatant
was transferred to a fresh tube. The sample was
The homogenized samples were incubated for 5 stored at -5 to -20°C for future use.
minutes at 15-30°C. An addition of 0.2 chloroform
per 1 mL of TRIZOL® Reagent was done, followed 2.8 Construction of Standard Calibration Curve
by shaking the tubes vigorously by hand for 15
seconds. The tubes were then incubated at 15-30°C The solutions in Table 1 below were prepared in 1.5
for 2 to 3 minutes. Afterwards, the samples were mL MCTs. An aliquot of 300 μL of each solution
centrifuged at no more than 12000×g for 15 minutes were transferred to 1.5 mL plastic cuvettes. Addition
at 2 to 8°C. the mixture then separated into (1) a of 1200 μL Bradford reagent was then done and the
lower red phenol-chloroform phase, (2) an solution was pipette-mixed.
interphase, and (3) a colorless upper aqueous
phase. The aqueous phase was discarded. Table 1. Volumes of BSA, urea, and diH​2​O to be added for
the preparation of standard solutions.
2.4 DNA Precipitation Standard Vol BSA Vol 10 M Vol Sterile
No. solution (μL) Urea (μL) diH2​ ​O (μL)
For the lower red phenol-chloroform phase and Blank 0 2 998
interphase, 0.3 mL 100% ethanol was added per 1 1 10 2 988
mL of TRIZOL® Reagent used in the initial 2 20 2 978
homogenization. The samples were mixed by 3 30 2 968
inversion. After, they were stored at 15-30°C for 2 to
4 40 2 958
3 minutes. The DNA was precipitated by centrifuging
5 50 2 948
at no more than 2000×g for 5 minutes at 2-8°C. the
pellet was discarded, and the phenol-ethanol 6 60 2 938
supernatant was saved. 7 70 2 928
8 80 2 918
2.5 Protein Precipitation 9 90 2 908
10 100 2 898
The supernatant was added with 1.5 mL 100%
isopropanol per 1 mL TRIZOL® Reagent used for

Biochemistry 124.1 Total Protein Isolation and Quantification ​Page 2 of 7

A replicate of the blank standard was also prepared.
This served as the reference for the UV-Vis analysis.
The λ​max of standard no. 10 was first determined. Its
values were assured to be equal to 595 ± 5 nm. The
absorbance values of the standards were then
obtained from the λ​max​. Finally, a standard curve of
the linear equation was constructed.

2.9 Real Sample Quantitation

The protein extraction solution obtained in 2.7 was

diluted 10-fold by adding 900 μL deionized water to
100 μL of the sample. The solution was vortexed at Figure 1. ​Bradford assay standard curve generated using
1mg/mL of BSA.
low speed for homogenization.
The resulting standard curve gives an equation of
An aliquot of 300 μL of the solution was transferred
the line:
to 1.5 mL plastic cuvette and 1200 μL of Bradford
y = 13.58x + 0.3736
reagent was added. The solution was pipette-mixed.
Two other aliquots were done to have samples in where
triplicate. From the standard curve, the concentration y = absorbance
of the diluted sample was determined. The dilution x = concentration
factor involved was considered and the original
concentration of the sample was calculated. With that, the absorbances of the different mongo
total protein samples and their corresponding
3 RESULTS concentrations are shown in Table 2.

Table 3. ​Absorbances and concentration of mongo

After the isolation of proteins from different growing
samples.
stages of mongo beans, quantification was done Concentration
through Bradford assay. Generation of a standard Sample Absorbance
(mg/mL)
curve was primarily done to be able to calculate the Seeds 0.2284 -0.0107
concentration of the protein samples. 1-2 Days 0.368 -4.124 x10​-4
3-4 Days 0.2967 -5.051 x10​-3
Table 2.​ Averaged absorbance readings of standards.
Concentration of Absorbance 4 DISCUSSION
Standard No.
BSA (mg/mL) (Ave.)
Blank 0 0 Total protein isolation from a specific sample
1 0.01 0.492 includes three main processes: homogenization,
2 0.02 0.82 precipitation, and solubilization. The first involves the
use of mechanical and chemical processes to break
3 0.03 1.0575
down the sample into a homogenized mix. The
4 0.04 1.0795 second involves the addition of chemical reagents to
5 0.05 1.196 isolate and retrieve the total protein samples. The
6 0.06 1.2105 third involves the use of an appropriate buffer
7 0.07 1.221 solution to re-dissolve the total protein sample for
8 0.08 1.177 future storage and use.
9 0.09 1.248
10 0.10 2.077 4.1 Protein Isolation

For the homogenization, the mongo plant samples

were placed with dry ice to allow freezing and easier
homogenization. Grinding took place afterwards.
After the physical grinding, TRIZOL® reagent was
added to allow for cell disruption and dissolution of
the cell components. Insoluble materials formed
which include carbohydrates, cellular organelles,
and big DNA fragments were then centrifuged

Biochemistry 124.1 Total Protein Isolation and Quantification ​Page 3 of 7

leaving the supernatant containing the mongo RNA, minute concentrations. Although one downside here
DNA, and proteins (Tan & Yiap, 2009). is the possibility of salts being incorporated into the
precipitate due to the usage of chaotropic salts (Tan
The TRIZOL® reagent contains phenol, and & Yiap, 2009).
guanidine isothiocyanate. These two allow for the
sequential isolation of a plant's RNA, DNA, and total The end-product of the isolation procedure is the
proteins. Guanidine isothiocyanate acts to protect total protein of the sample. Although the goal of the
RNA from nuclease reactions meanwhile it also acts isolation 100% yield of the protein being examined,
as a detergent for breaking down the cellular walls this is almost always not the case. What is observed
and membranes of the sample (Chomczynski & are percentage yields of around 47% for example in
Sacchi, 2006). one study (Wong et al., 2016). As such, the actual
yields are always lower than the expected yield. For
After the isolation of the supernatant, chloroform is the extraction process, it is highly encouraged to aim
added to the solution. Chloroform functions by for higher yield than purity.
adding a density gradient in the solution (Tan &
Yiap, 2009). This allows for the formation of a In relation to yields another parameter that can be
three-phase solution. A red phenol-chloroform phase measured during the protein isolation procedure is
and the interphase contains the DNA and proteins the specific activity of a protein. This parameter is
while the RNA is found in the colorless upper used as a measure of protein purity in a purification
aqueous layer. The latter is then discarded after or isolation setup. Specific activity is the measure of
centrifugation, and 100% ethanol is added to the the amount of a specific enzymatic product given a
remaining layers. The solution was then centrifuged specific weight of sample (Specific Activity, n.d.).
to separate the DNA precipitate from the dissolved The total protein can be further separated and
proteins. isolated using separation techniques such as 2D
SDS-PAGE and affinity chromatography. These
The addition of ethanol to the solution allows for the methods enable to isolate specific sets of proteins
precipitation of the DNA into a pellet leaving the that would otherwise be difficult to analyze in a total
phenol-ethanol supernatant containing the total protein samples.
protein sample. Ethanol works to lower the dielectric
constant of the solution which forces the DNA to 4.2 Protein Quantification
come together and precipitate out of the solution. To
the remaining supernatant, isopropanol was added After obtaining the total protein samples,
to precipitate the proteins from solution. The solution quantification methods can then be applied to
was then centrifuged with the precipitate isolated. determine how much protein sample was obtained
The use of isopropanol is similar to that with the during the procedure. Many methods of protein
addition of ethanol to precipitate the proteins (Egusa quantification have been developed such as the
et al., 1983) Lowry assay, Bicinchoninic assay, and the Bradford
assay. In the experiment, the lattermost was the
To this precipitate, guanidine hydrochloride was choice of assay in determining the total protein
added with 95% ethanol. This was done to remove quantity present in the mongo seeds.
the phenols found within the precipitate. After three
subsequent washes and supernatant removal, the The Bradford assay is best used for instances
resulting protein pellet was washed with ethanol and wherein the total protein samples are small in
air dried for 15 minutes. The pellet was resuspended concentrations of up to 2 mg/mL. The Bradford
in 0.1% SDS. This reagent functions as a solvent assay relies on the complexation reaction of the
containing both polar and nonpolar parts that help in Coomassie Blue G-250 with specific residues in the
solubilizing the protein pellet into solution (Likhite & peptide chain which results in a shift of absorbance
Warawdekar, 2011). depending on the presence of acidic or basic
residues. The differing absorbances on a specific
For the extraction of proteins from fluids, such as wavelength, 595 nm for basic residues, can then be
those coming from animals, centrifugation in a high monitored using a spectrophotometer to determine
density medium must be done to isolate the cell in the quantity of protein present in a given sample
question before proceeding with the lysis, (Bradford Reagent, n.d.).
precipitation, and solubilization stages. Precipitation
using the salting out method is highly in focus to For the assay procedures, standards were first
allow the extraction of proteins, especially that of prepared. The Bradford assay standards used

Biochemistry 124.1 Total Protein Isolation and Quantification ​Page 4 of 7

comprised of bovine serum albumin (BSA), 10 M It is therefore recommended to generate a standard
urea, and ddH​2​O. BSA is used as the protein curve with better linearity. Furthermore, addition of
standard due to its stability, colorless nature and polyvinylpolypyrrolidone (PVPP) to TRIZOL®
ability to react with CBG readily. Meanwhile, urea is method would theoretically reduce protein
added to function as a denaturing reagent to ensure degradation by phenolics (Wang et al., 2008) thus
all proteins are denatured to allow for accurate increasing product yield.
absorbance measurement (Robinson & Jencks,
1965). REFERENCES

Total protein yields extracted from plants Bonjoch, N. P., & Tamayo, P. R. (2001). Protein
theoretically should decrease with the plant's age. content quantification by Bradford method.
The seeds of plants provide the highest protein In ​Handbook of Plant Ecophysiology
yields while a four year old plant will have a marked Techniques ​(pp. 283-295). Netherlands:
decrease in total protein yield. The process of Kluwer Academic.
protein extraction in mature plants is more difficult
due to the presence of a thicker cell wall which is Boyer, R. (2000). ​Modern Experimental Biochemistry
harder to disrupt. Add to this the presence of more (3rd ed.). San Francisco, CA: Addison
nonprotein components which are more difficult to Wesley Longman.
remove such as polyphenols and terpenes. (Islam et
al, 2004). Bradford reagent. (n.d.). Retreived from
https://www.sigmaaldrich.com/content/dam/s
The concentration values obtained were of negative igma-aldrich/docs/Sigma/Bulletin/b6916bul.p
value due to a lower actual protein concentration df
than the standards used for the linear regression. ​A
possible source of error in the protein isolation may Chomczynski, P. (1993). A reagent for the
also originate from incomplete solubilization of the single-step simultaneous isolation of RNA,
protein pellet which may lower total yields obtained. DNA and protein from cell and tissue
Another source of error may come from samples. ​Biotechniques, 15​(3), 532-534.
contamination of the protein pellet that may yield a Retrieved from National Center for
positive error on the absorbance reading and Biotechnology Information (NCBI) website:
subsequently the protein concentration (TRIzol​® https://www.ncbi.nlm.nih.gov/pubmed/76928
Reagent, n.d.) 96

5 CONCLUSION & RECOMMENDATIONS Chomczynski, P., & Sacchi, N. (2006). The

single-step method of RNA isolation by acid
Essentially, the procedure was composed of protein guanidinium thiocyanate–phenol–chloroform
isolation and a sequential quantification of different extraction: twenty-something years on.
growing stages of mongo beans. It was found that Nature Protocols, 1(2), 581–585.
proteins can be successfully extracted from seed, doi:10.1038/nprot.2006.83
two-day old sprout, and four-day old sprout using
TRIZOL®. The protein content was also successfully Egusa, G., Brady, D. W., Grundy, S. M., & Howard,
quantified using Bradford assay. Using a standard B. V. (1983). Isopropanol precipitation
curve of R​2 of 0.7643, the concentration of seed, method for the determination of
two-day old sprout, and four-day old sprout were apolipoprotein B specific activity and plasma
calculated to be -0.0107, -4.124 x10​-4​, and -5.051 concentrations during metabolic studies of
x10​-3 mg/mL, respectively. The negative values in very low density lipoprotein and low density
the concentration obtain is due to a lower actual lipoprotein apolipoprotein B. Journal of lipid
protein concentration than the standards used for research, 24(9), 1261-1267.
the linear regression. Theoretically, protein levels
should decrease as the beans age. This is due to Islam, N., Lonsdale, M., Upadhyaya, N. M., Higgins,
the fact that more nonprotein components develop T. J., Hirano, H., & Akhurst, R. (2004).
as plants mature. Possible sources of error in the Protein extraction from mature rice leaves
isolation procedure include protein pellet for two-dimensional gel electrophoresis and
contamination and incomplete solubilization of the its application in proteome analysis.
protein precipitate. Proteomics,​ ​4​(7), 1903-1908.

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Likhite, N., & Warawdekar, U. M. (2011). A unique Present. Journal of Biomedicine and
method for isolation and solubilization. Biotechnology, 2009, 1–10.
Journal of Biomolecular Techniques, 22​(1), doi:10.1155/2009/574398
37-44. Retrieved from National Center for
Biotechnology Information (NCBI) website: Trizol reagent. (n.d.). Retrieved from
https://www.ncbi.nlm.nih.gov/pmc/articles/P https://assets.thermofisher.com/TFS-Assets/
MC3059540/ LSG/manuals/trizol_reagent.pdf

Robinson, D. R., & Jencks, W. P. (1965). The effect Wang, W., Tai, F., & Chen, S. (2008). Optimizing
of compounds of the urea-guanidinium class protein extraction from plant tissues for
on the activity coefficient of enhanced proteomics analysis. Journal of
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compounds1. ​Journal of the American 10.1002/jssc.200800087
Chemical Society,​ ​87(​ 11), 2462-2470.
Wong, S. Y., Lee, C. C., Ashrafzadeh, A., Junit, S.
Specific activity of enzyme solutions. (n.d.). M., Abrahim, N., & Hashim, O. H. (2016). A
Retreived from High-Yield Two-Hour Protocol for Extraction
https://www4.uwsp.edu/chemistry/tzamis/36 of Human Hair Shaft Proteins. PLOS ONE,
5f00pdfs/specificactivity10.pdf 11(10), e0164993.
doi:10.1371/journal.pone.0164993
Tan, S. C., & Yiap, B. C. (2009). DNA, RNA, and
Protein Extraction: The Past and The

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APPENDIX
1.1 Raw Data

Table A.​ Absorbance values of standard solutions, prepared in duplicates.

Standard Absorbance Trial #1 Absorbance Trial #2 Averaged Absorbance
Blank 0 0 0
1 0.395 0.589 0.492
2 0.814 0.826 0.82
3 1.022 1.093 1.0575
4 0.947 1.212 1.0795
5 1.121 1.271 1.196
6 1.073 1.348 1.2105
7 1.115 1.327 1.221
8 1.03 1.324 1.177
9 1.117 1.379 1.248
10 1.144 3.01 2.077

Table B. ​Absorbance values of protein samples from different growth stages of mongo beans, prepared in duplicates..
SEEDS 1-2 DAYS SPROUT 3-4 DAYS SPROUT

Jung et al. Jung et al. Gutierrez et Belber et al. Isidro et al. Cano et al. Lazaro et Ora et al.
al. al.
R1 0.605 0.304 0.241 0.393 0.351 0.219 0.337 0.270
R2 0.402 0.312 0.192 0.422 0.333 0.242 0.402 0.310
R3 0.341
Average 0.2284 0.368 0.2967

1.2 Sample Calculations

For the determination of the standard concentration (using Standard No. 1):
M 1V 1 = M 2V 2
M V
[BSA] = M 2 = V1 1
2
1 μg
( )(10 μL)
[BSA] = μL1000 μL
10 μg
[BSA] = 1000 μL
μg
[BSA] = 0.01 μL

For the determination of the protein concentration (using seed sample):

Given the equation of the standard curve: y = 13.58x + 0.3736
where y = A = 0.2284

0.2284−0.3736
[seed ptn] = x = 13.58
mg
[seed ptn] = − 0.0107 mL

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