Elissia Vecere

Section 102
3/17/2021
Protein Electrophoresis
Introduction
SDS-polyacrylamide gel electrophoresis can be used in order to separate proteins based upon
the molecular weight of the proteins. Throughout the experiment, the student makes use of a
gel apparatus, where proteins will be loaded into the lanes in presence of an electric current via
a cathode. The lab works in the sense that the proteins present are negatively charged.
Therefore, by running a current throughout the gel slab, the proteins were travel from the
cathode, where positively charged atoms are attracted, due to its negative nature to the anode,
where negatively charged atoms are attracted, due to its positive nature. The different bands
that will be made present are due to the difference in sizes of the proteins. As a result of
proteins being smaller, they will overall, travel faster, and as a result of proteins being larger,
they will travel slower within the gel plate. This can be observed by proper completion of this
lab.

Objectives
The objectives for this lab consisted of the student gaining the ability to understand the
concepts of basic polyacrylamide electrophoresis, including why different percentages of
acrylamide are used and by what mechanism the proteins are separated. The student should
also understand the use of SDS at the end of the lab, along with being able to calculate the
approximate molecular weight of proteins separated by electrophoresis.

Procedure
Preparation of Protein Samples for Electrophoresis
The student began the experiment by weighing the dried protein preparations of casein
and albumin from the prior in person lab in order to complete that lab report. Following this,
the student obtained four Eppendorf tubes in order to create 2 tubes of the 2 mg/mL stock
solution and 2 tubes for the gel samples. Casein and albumin samples were then made in two of
the Eppendorf tubes by dissolving approximately 2 mg of each purified protein with 1000
microliters od distilled water in separate tubes. This provided the student with the 2 stock
solutions mentioned prior, with concentrations of 2 mg/mL. Next, the student added 10
microliters of each purified stock samples into two different tubes (keeping casein and albumin
solutions in their separate containers), along with 20 microliters of water and 10 microliters of
sample buffer. The total volume was 40 microliters in the two tubes. Next, the solutions were
centrifuged and then heated by a heating block at 95 degrees Celsius was used to heat the
solutions for 5 minutes. Finally, the solutions were centrifuged again.
Analysis of Purified Protein by Electrophoresis
To begin this portion of the experiment, the student observed the TA prepare a 10%
PAGE gel plate. Running buffer was poured into the upper chamber to check for leaks. Then
running buffer was poured into the lower chambers in order to reach the bottom edge of the
gel. The combs of the gel were gently pulled in order to allow the buffer to flow into the wells.
With a micropipette the student loaded 15 microliters of their samples (milk and egg rotation
for each lane) into the wells of the gel. Then once all of the samples were loaded, the top of the
cell was put on and the leads were connected to the power supply. The voltage was set to 170
 Elissia Vecere
Section 102
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Protein Electrophoresis
volts. The observations of the gel were recorded at 0 minutes, 15 minutes, and about 30
minutes. Afterwards, the power source was removed from the tracking gel and the gel plate
was removed. The TA continued on in completing the staining procedure by using Gelcode Blue
stain and allowed that to sit with the gel for approximately 15 minutes, after heating it for 60
seconds two times. After the 15 minutes were up, the gel was rinsed with DI water and sat for
another 15 minutes. Images were taken of the final product.

Observations
The student noticed that the buffer solution added had a purple/blue color. Afterwards,
once the voltmeter was attached to allow for electrophoresis to begin, bubbles began coming
out. This was a sign to show that the device was working, and the volts were running. At zero
minutes, there was no change and the dyed solution remained sitting at the top of the “boxes”.
After 15 minutes, the solutions moved relatively lower down, and kind of in line with each
other. However, one newly observed thing at this time for the student was the presence of blue
and yellow bands within all of them. After 30 minutes had passed, the blue and yellow bands
had moved further down, towards the positively charged anode. There also was a very clear
ladder present in the first row. Finally, after heating and staining the gel, it sat for 15 more
minutes. Once this portion was completed, the student was able to see the individual bands on
the gel like block.

Results
Tables and graph below represent an answer for number 1 of the post lab assignment.
Calculations were completed in the lab notebook.

Marker
Distance Migrated (cm) Log (Molecular Weight) Molecular Weight (kDa)

1.9 2.278 190

2.4 2.060 115

2.7 1.903 80
3.3 1.845 70

4.1 1.699 50

4.9 1.477 30
5.3 1.398 25
6.8 1.176 15
8 1.00 10
 Elissia Vecere
Section 102
3/17/2021
Protein Electrophoresis

Marker Standard Curve

2.5
Log (Molecular Weight) kDa

2 f(x) = − 0.199924011171007 x + 2.52366733779308

1.5

0.5

0
1 2 3 4 5 6 7 8 9
Distance Migrated (cm)

Albumin
Distance from Loading Well to Band (cm) Calculated Molecular Weight (kDa)
2.0 133.01
2.8 92.04
3.1 80.17
3.6 63.70
4.8 36.64

Casein
Distance from Loading Well to Band (cm) Calculated Molecular Weight (kDa)
2.1 127.03
2.6 100.93
3.4 69.82
4.8 36.63

Purified Albumin
Distance from Loading Well to Band (cm) Calculated Molecular Weight (kDa)
3.1 80.17
3.3 73.11
4.4 44.06
4.8 36.64

Discussion

2. If more than one band appears within the sample, it means that more than one protein is
present and therefore, the sample is not completely pure. The presence of multiple bands could
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Section 102
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Protein Electrophoresis
also indicate proteolytic degradation. Finally, the presence of a double band can indicate two
polypeptides that share amino acid sequences that are similar in structure but have minor
differences.

3. SDS-Page is used in order to separate proteins from each other based on their size.
Specifically, smaller proteins will travel further on the gel plate due to their ability to move
faster. Larger proteins will move slower and will therefore travel a shorter distance on the gel
plate. To begin, SDS-Page is known as Sodium Dodecyl Sulfate-Polyacrylamide Gel
Electrophoresis. The system consists of one gel with another being poured on top of it. The
teeth of the combs within the main gels creates the wells of the gel. Here, the protein solution
can be inserted into the wells. At this point, the protein solution has already first been
completely denatured and mixed with sodium dodecyl sulfate in order to give all of the proteins
a negative charge. This formation allows for the properties of the proteins to be kept constant,
ensuring that their differences will only vary in their sizes. When the system is attached to a
charge, the negatively charged protein present in the wells will travel from the cathode, also
negatively charged, toward the anode, which is positively charged. Throughout the traveling
process, the larger proteins will encounter more resistance when moving through the gels and
will represent bands closer to the cathode. The smaller proteins will move through the holes of
the gels more easily and will show bands closer to the anode.

4. The types of proteins found in eggs, excluding albumin, include ovomucins, lysozyme,
ovotransferrin and ovomucoid (Aberyathne et al. 2019).

CITATION

Abeyrathne, E.D.N.S., et al. “Egg White Proteins and Their Potential Use in Food Processing or
as Nutraceutical and Pharmaceutical Agents-A Review.” Poultry Science, Elsevier, 11
Dec. 2019, www.sciencedirect.com/science/article/pii/S0032579119384500. 

Conclusion
Throughout this lab, the student grew an understanding for basic polyacrylamide
electrophoresis, the use of SDS, and how to calculate approximate molecular weight of proteins
via the use of electrophoresis. More specifically, the student was able to work directly with
electrophoresis to observe the movements of the proteins within a gel plate when a cathode,
anode, and current were present. With the use of prior derived albumin and casein from milk
and eggs, the student was able to make a 40 mL solution of each individual proteins, along with
a buffer and water as a dilutant and observe its movement within electrophoresis. This
movement was then able to be used in order to determine the molecular weight markers that
were ran throughout the gel. Overall, the lab allowed for the student to observe individual
molecular weights throughout the proteins present, along with understanding the depths of the
proteins in food we commonly consume.
 Elissia Vecere
Section 102
3/17/2021
Protein Electrophoresis