Review feedback from your instructor (in Grades) on your final draft of Writing Project 1 and/or the half draft of this project, from your peers (in M05 Peer Response–Writing Project….
Describe the basic steps involved in protein digestion, protein denaturation, and electrophoresis
How “Chicken” Are You?
Using peptide mapping to determine amino acid sequence similarities1
After this lab exercise you should be able to:
- Describe the basic steps involved in protein digestion, protein denaturation, and
- Explain how SDS gel electrophoresis works to separate polypeptide pieces based on
- Describe the importance of SDS and the reducing agent to this method.
- Explain the role of the tracking dye, the stain, and molecular markers in
- Describe what proteases are and list some examples of their practical uses.
- Explain how this method may be used to determine structural relatedness of a protein
in different species, and perhaps evolutionary relationships among species.
- Explain the limitations of this type of study.
Before the lab review the following topics in your textbook
• The structure of proteins and denaturation and sequence homology (section 3.4)
• Gel electrophoresis
As you go through the lab, answer the questions in the handout. You will submit the
questions that are assigned point values as your assignment for this lab. The assignment
is due before the beginning of lab 6.
Proteins carry out many different functions in living cells. They can be structural
components, enzymes that carry out specific reactions, receptors that receive external
signals, etc. The function of a protein depends on its three-dimensional shape, which in
turn depends on the protein’s primary structure = its amino acid sequence. When the
protein loses its three-dimensional shape = it denatures, it will also lose its function.
After a polypeptide is synthesized in the cell, it has to be folded into its proper shape.
This involves the formation of bonds that lead to the proper folding of the polypeptide
into secondary and tertiary structures. The folding of the polypeptide into the secondary
structure of the protein backbone such as an -helix or a -pleated sheet involves the
formation of weak hydrogen bonds. These bonds can be easily broken by heat or changes
in pH, for example. The overall three-dimensional structure of the polypeptide is the
tertiary structure. Tertiary structure is formed by interactions among amino acid side
chains and involves weak interactions including hydrophobic / hydrophilic interactions
and hydrogen bonds, ionic bonds, and covalent bonds such as disulfide bridges formed
between two cysteine residues. If the protein contains more than one polypeptide, the
structure is referred to as quaternary structure.
1 This exercise uses a kit from Modern Biology Inc.
Proteins that carry out essential functions for the cell tend to change very little in a
population through mutations over time. That means their amino acid sequences are
highly conserved. Therefore, we can look at the amino acid sequences for the same
protein from different species, and we can use the level of similarities and differences
among different species to determine how closely related they are to each other; that is,
we can assess their evolutionary relationships based on these sequences. Please review
the case of cytochrome c described in your textbook (section 3.4) as one such example.
Proteases are enzymes that cut (cleave) polypeptides at specific amino acid sequences.
The digestive enzyme chymotrypsin, for example, cuts peptide bonds formed from the
carbonyl groups of tryptophan, phenylalanine, and tyrosine. Another protease, papain
(derived from Papaya) cuts peptide bonds formed from leucine and glycine. Thus,
depending on which enzyme is used, and where the target amino acids are located, a
polypeptide could be cut to generate different size fragments. As an example, consider
the polypeptides in Figure 1 below. They all have the same starting length. They are
almost identical in molecular weight. The potential cut sites for the enzyme
chymotrypsin have been marked.
Protein 1: val glu glu gln arg trp val leu ala his thr gln arg glu gln met val ala
Protein 2: val glu glu gln arg trp val leu ala his thr gln phe glu gln met val ala
Protein 3: val thr gln phe glu leu arg glu trp val ala met gln phe gln glu val ala
Figure 1: The amino acid sequences for three small proteins. Arrows mark the potential
cut sites for the enzyme chymotrypsin.
How many fragments would be produced by cutting these small proteins with
chymotrypsin? Will any of the fragments be identical in size among the three proteins?
If polypeptides from closely related species have the same amino acid sequence with the
same amino acid residues at the same location, then treatment with the same enzyme
should lead to synthesis of similar length fragments. Now consider a mutation in one
species alters one of the amino acids that is the target of the enzyme so that a cut site is
lost due to the mutation, or a new cut site is formed. In this case, it is likely that when the
polypeptide extracted from this species is treated with the same enzyme, different size
fragments will be formed compared to other related species without such a mutation. As
an example, compare proteins 1 and 2 in Figure 1 above. The pattern of fragments
generated can be used to determine how closely related different species are based on
amino acid sequence similarities for a given polypeptide. For this approach to work,
these peptide fragments have to be separated according to their sizes.
One common method to separate peptide fragments based on size is SDS gel
electrophoresis. You can learn about gel electrophoresis and its use in separating DNA
fragments by reading chapters 14 and 17 in your textbook. The method used to separate
peptide fragments is similar in principle. An electric field is applied to the fragments,
which will then move away from the negative electrode towards the positive electrode.
DNA naturally has negative charges (why?). The electric charge of polypeptides depends
on the ionization of the amino acids, which itself is dependent on the pH of the solution.
Peptide fragments thus have to be coated with uniform charges before gel electrophoresis
can work. An agarose or polyacrylamide gel is typically used as the medium through
which the fragments move. The gel is made with depressions (wells) in which the
fragments are loaded before the electric field is applied. When the electric field is
applied, the fragments then have to move through the gel from the negative to the
positive poles. The gel serves as a “sieve” through which the fragments have to pass.
The larger the fragment is, the more slowly it will move through the “sieve”, and thus gel
electrophoresis allows us to separate the fragments based on size. Molecular markers
(peptide fragments) of known size, also called a ladder, are included in the procedure for
comparison with the fragments of interest. The gel is stained with some sort of stain to
visualize the fragments. The locations of the fragments on the gel can be compared with
those of the fragments in the ladder that have also passed through the same gel with the
same electric field in order to determine the size of the fragments of interest.
For this method to work with polypeptides, they have to be denatured and linearized.
Any disulfide bridges have to be cut to help linearize the polypeptides. This is carried
out by boiling the samples in the presence of reducing agents, such as -mercaptoethanol,
as well as the anionic detergent SDS (sodium dodecyl sulfate). The detergent SDS also
covers the native charge of the amino acids in polypeptides with its own negative
charges. Thus, any peptide fragment will be linear and uniformly negatively charged.
Therefore, neither the original three dimensional shape nor the charges of ionizable
amino acids will interfere with the movement of the fragments through the gel. The only
thing that will matter will be the size of the fragment. Since the pH of the solution
matters as far as ionization of amino acids is concerned, the pH of the solution (buffer)
used during electrophoresis is important to make sure the denatured polypeptides are
uniformly negatively charged.
5 points: As an example, consider albumin, a protein made of a single polypeptide
weighing 66,000 daltons (66 kDa). On the other hand, gamma globulin has quaternary
structure: is made of four polypeptides, two of which weigh 23,000 daltons (23 kDa)
each, and two of which weigh 53,000 daltons (53 kDa) each. When treated with reducing
agent and SDS, the subunits separate and they all linearize. If albumin and gamma
globulin were run through gel electrophoresis, which polypeptides would move the
fastest? Which would move the slowest?
10 points: Consider the proteins in Figure 1. Assume they are treated with chymotrypsin
to cut them into fragments, and then the fragments are separated by gel electrophoresis.
What would the fragment patterns look like in the gel for the three different proteins,
assuming we can separate polypeptides that differ in size by very small amounts?
Complete Figure 2 below to show the location of the uncut polypeptides (- enzyme lanes)
and the cut fragments (+ enzyme lanes).
Figure 2: Gel electrophoresis of proteins from Figure 1 (- enzyme lanes) as well as cut
fragments from those proteins generated through treatment by chymotrypsin (+ enzyme
lanes). The location of the band for the uncut protein 1 is shown.
In this experiment we will be comparing the structural similarity of serum albumin from
several different vertebrate species and thus examining how closely related the proteins
from these species are to each other. These proteins have similar molecular weights. We
will first cut the proteins with the enzyme chymotrypsin (enzyme digest). We will then
separate the fragments based on their relative sizes through gel electrophoresis so that we
can compare the fragments generated by the enzyme digest for the different species.
4 points: Your instructor will let you know which species will be studied in your lab.
Based on that, write the hypothesis and prediction for the experiment.
• Serum albumin from several different vertebrate species
• Sample buffer containing glycerol, SDS, -mercaptoethanol, and bromphenol blue
• Coomassie blue stain (40% methanol, 10% glacial acetic acid, 0.25% Coomassie
brilliant blue R-250)
• De-staining solution (20% methanol, 5% glacial acetic acid)
• Electrophoresis buffer
• Standard protein markers (BioLabs P7706S/L)
• Incubator at 37C
• Ice bath
• Boiling water bath
• Bench-top shaker
• Gel electrophoresis chambers
• 5% agarose gels
• Micropipettors and tips
• Gloves and goggles
Part I: Enzyme digest
- Wear goggles and gloves.
- Each group will be given the serum albumin from one species. For your group, label
3 microcentrifuge tubes 1, 2, and 3.
- To each microcentrifuge tube add 10 μL serum albumin for the species assigned to
- Add 10 μL distilled water to tube 1 and 10 μL chymotrypsin to each of tubes 2 and 3.
- Add 20 μL sample buffer to tube 1, mix and place in an ice bath.
- Place tubes 2 and 3 in the 37C incubator. Set a timer for 5 minutes for tube 2. Set
another timer for 30 minutes for tube 3.
- Remove tube 2 from the incubator after 5 minutes. Add 20 μL sample buffer to tube
2, mix and place in an ice bath.
- Remove tube 3 from the incubator after 30 minutes. Add 20 μL sample buffer to tube
3 and mix.
- Mix the contents of each tube well before boiling the samples.
- Either use cap locks for the microcentrifuge tubes, or use a straight pin to pierce the
lid of the tubes to provide a vent for steam to escape the tubes during boiling. Care
should be taken to make sure the tubes are not inverted in the water bath so that the
contents are not lost or diluted!
- Once the water is boiling vigorously, place the tubes in a rack and place the rack in
the boiling water bath for 5 minutes.
Part II: Analyzing peptide fragments by gel electrophoresis
- Your instructor will prepare the gel and place the gel into the electrophoresis chamber
and will add enough buffer to cover the surface of the gel.
- Practice loading mock samples (dye solution) into the wells in a practice gel. You
need to be comfortable with inserting the micropipettor tip into a well without
pushing too far to puncture the bottom of the well. You also need to be comfortable
injecting the sample into the well and removing the micropipettor tip without sucking
the sample back into the pipette or releasing the sample into the buffer solution
instead of the well.
- One gel will be used to analyze the peptide fragments from the tubes for 2 groups.
Depending on the number of groups, the serum albumin from one or two species may
be analyzed by more than one group.
- Follow Table 1 below to load 10 μL of the samples/markers into each well of a gel.
Table 1: The order of samples loaded into the wells of the gel for electrophoresis.
Sample well # Sample
1 Standard proteins (ladder)
2 Protein sample from Group 1 Tube 1
3 Protein sample from Group 1 Tube 2
4 Protein sample from Group 1 Tube 3
5 Standard proteins (ladder)
6 Protein sample from Group 2 Tube 1
7 Protein sample from Group 2 Tube 2
8 Protein sample from Group 2 Tube 3
- 5 points: After all of the samples are loaded, connect the electrodes to the gel box.
Where must the negative electrode be in relation to the wells in the gel? Why?
- Turn on the power supply and run the gel at 100 V until the blue dye has migrated to
within 1 cm of the positive electrode end of the gel (approximately 3-4 hours).
- Turn off the power supply. Disconnect the electrical leads.
- Remove the gel from the unit and transfer to a staining box.
- The staining solution contains Coomassie blue stain as well as acetic acid. The acid
will precipitate and immobilize the proteins in the structure of the gel matrix so that
the protein bands do not become blurry due to diffusion. Cover the gel with staining
solution and allow it to stand for 1-2 hours.
- After the staining step, the unbound stain must be removed from the gel. This is done
through the de-staining step. The de-staining solution is a dilute mixture of acetic
acid and methanol. Pour out the extra stain (can be saved for future use) and add destaining
solution to cover the gel. De-stain for 4 hours with gentle shaking.
- Examine the gel to observe the bands. Record the relative migration of the standards
and the bands observed in the lanes for tubes 1-3 for each species studied.
- Interpret each lane of the gel:
- 6 points: What do you expect in the lane for #1 tubes? Explain the reason for your
expectation. Do the results match the expectations?
- 5 points: Do you observe differences in the lanes for tubes 2 and 3? What does that
- 10 points: Are there differences among the species? What does that tell you about
how closely related the proteins are from these species? Do the results match the
prediction and support the hypothesis?
- 5 points: How would your results have been different if an enzyme with a different
amino acid sequence preference had been used?