Activity 4.1.3 Protein Purification
Introduction
Thanks to you (and DNA derived from a jellyfish), tiny bacteria now glow a brilliant green under
UV light. You transferred a gene from one organism to another and you now see, in living color,
the relationship between DNA and a trait. But your work is not complete. You now have a
source of GFP, but the protein is trapped inside of the cells. Once scientists have used a
bacterial cell to produce a protein, such as insulin or GFP, how can they extract and isolate the
protein so it can be used? Bacteria produce thousands of different proteins. Scientists must
have a way of sifting through the mix and finding the “needle in the haystack.” As you continue
Lesson 4.1, your job is to separate the GFP from the other bacterial proteins, verify its purity,
and get it out to market.
Before protein products can be released as pharmaceuticals, enzymes, or diagnostic tools, they
must be isolated and purified in a series of laboratory procedures. Chromatography is one
technique used to separate and purify components in a mixture of gases, liquids, or dissolved
solids. In all chromatography methods, components are separated using a stationary phase and
a mobile phase. The stationary phase is the medium that does not move; the mobile phase is
the medium that does. A mixture is dissolved in the mobile phase, which may be gas or liquid,
and the sample is then passed over a stationary phase, a filter such as paper, silica on a glass
plate, or porous beads in a column. Just as a filter catches some materials and lets others slide
through, the stationary phase either traps or releases items in the mixture based on their
chemical and physical properties. This filter may separate out components in the mixture by
size, by charge or by their affinity for the filter itself. Specific molecules, such as proteins, can be
isolated as they are separated by chromatography.
Once your bacteria have multiplied in liquid culture, you will perform a specific type of
chromatography called hydrophobic interaction chromatography (HIC) to separate the GFP from
the other proteins in the mixture. Proteins have a unique structure based on the sequence of
their amino acids. GFP is a highly hydrophobic molecule because of its many water-fearing
amino acids. Using a column filled with porous beads and a series of buffer solutions, the
hydrophobic GFP proteins can be trapped in the beads of the hydrophobic column and then
washed out and collected. Follow the glowing drops and isolate our protein of interest!
Equipment
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Bio-Rad Green Fluorescent Protein (GFP) Purification Kit
o Disposable pipets
o Lysozyme
o Inoculation loops
o TE buffer
o HIC chromatography
o Binding buffer
columns
o Column equilibrium buffer
o LB broth with ampicillin and
o Collection tubes
arabinose
o Microcentrifuge tubes – 2ml
Computer with Internet access
 3D Molecular Designs – Amino Acid
Starter Kit
Laboratory journal
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Medical Interventions Activity 4.1.3 Protein Purification – Page 1
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Transformed bacteria on agar plates
from Activity 4.1.2
Activity 4.1.3 Student Response
Sheet
GFP Purification – Quick Guide
handout
Microcentrifuge
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Microcentrifuge tube rack
Handheld UV light
Freezer
Ring stand (optional)
Shaking incubator (optional)
Procedure
Part I: Review of Protein Structure
1. With a partner, brainstorm what you remember about protein structure. Think back to the
Designer Proteins activity you completed in PBS. Remember that a change in just one amino
acid of a protein can lead to an illness such as sickle cell disease. Take notes in your
laboratory journal.
2. Research the structure of an amino acid. Draw a generic amino acid in your laboratory
notebook and label each functional group.
3. Obtain an Amino Acid Starter Kit from your teacher.
4. Remove the magnetic chemical properties circle, the amino acid chart, and the magnetic
amino acid molecules from the kit.
5. Place each amino acid on the magnetic chemical properties circle according to its chemical
structure. Note that the different amino acid sidechains have been colored to reflect their
chemical properties.
6. Describe the distinguishing features of each category of amino acid side chain in the space
below the category. What do you notice about the molecules that make up this type of side
chain?
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Hydrophobic (nonpolar) side chains are YELLOW.
Hydrophilic (polar) side chains are WHITE.
Acidic side chains are RED.
Basic side chains are BLUE.
Cysteine side chains are GREEN.
7. Consult the amino acid chart to match each magnetic shape with the name of the amino acid
and to position it correctly on the magnetic circle.
8. Unroll a foam toober. This tube will represent the backbone of your protein.
9. Place a blue end cap on the N-terminus (the beginning) of the protein, and a red cap on the
C-terminus (the end) of the protein.
10. Evenly space 15 magnetic clips along the toober. Randomly add the following mixture of
amino acids by clipping the magnets on the amino acids to the magnets on the toober.
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6 hydrophobic amino acids
2 acidic amino acids
2 basic amino acids
2 amino acids containing cysteine
3 hydrophilic amino acids.
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Medical Interventions Activity 4.1.3 Protein Purification – Page 2
11. As a class, discuss how each of these amino acids will interact with one another and come
up with a list of “rules” for protein folding. Write these rules in your laboratory journal.
12. Fold your protein according to the rules described in your list.
13. Trade completed proteins with another group in the class.
14. Analyze the new protein to make sure that the amino acids are oriented in a way that
maintains the rules of protein folding. Provide feedback to the other group, but do not refold
the protein. Allow the group to make changes (if necessary) based on your observations.
15. Answer Conclusion questions 1-3.
Part II: Protein Purification by Column Chromatography
GFP is composed of many hydrophobic amino acids. This property can be used to separate the
protein from others in a mixture. In a high salt concentration, these hydrophobic amino acids
move to the outside of the protein and push the hydrophilic amino acids to the inside. The more
hydrophobic the compound, the more likely the compound will stick to a hydrophobic column.
When the salt concentration is reversed, the 3-D structure of the protein changes as the
hydrophobic amino acids move to the interior. Thus the protein is released from the beads in the
column and can be collected by itself.
16. Use the Internet to research the science of chromatography. Define the term
chromatography in your laboratory journal. Under the definition, list at least two uses of
chromatography in the real world.
17. Obtain a copy of the Bio-Rad GFP Purification – Quick Guide handout and a Student
Response Sheet from your teacher.
18. Read the entire procedure as described on the Quick Guide. Notice that there are four
lessons in the experiment. As Lesson 1 refers to the delivery of background information, you
will begin the experiment at Lesson 2.
19. Locate the name of each lesson of the experiment on the Student Response Sheet. Under
the name, summarize the overall goal of this specific part of the experiment.
20. Follow the steps of the Quick Guide and the instructions provided by your teacher to
complete the experiment. Use the drawings located on the right side of the page as a visual
reference for each step and make sure to follow the two bullets below. Your teacher will
inform you of appropriate stopping points. Record any observations in your laboratory
journal. The following additional instructions should also be followed. Note these changes on
the Quick Guide.
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In Lesson 4, step 4, transfer an additional 50µl of the supernatant to a clean
microcentrifuge tube labeled pre-purification supernatant. Label the tube with your initials
or lab designation and the date and place this tube in the designated freezer. You will use
this sample in the next experiment.
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In Lesson 5, you may be directed to use a ring stand to help stabilize your
chromatography column. Note that in this lesson, the columns are designed to drip
slowly. The entire process should take anywhere from 20-30 minutes. The column should
only be disturbed when gently changing out the collection tube. You may need additional
TE buffer to fully elute the glowing sample from the column. If so, label a new collection
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Medical Interventions Activity 4.1.3 Protein Purification – Page 3
tube as Tube 4. In increments of 50µl, add TE buffer to the column until all of the GFP
has been removed. Collect these drops in Tube 4.
21. At the conclusion of each part of the experiment, answer the associated questions on the
Student Response Sheet.
22. Discuss the answers to the questions with your lab group and with the class.
23. At the conclusion of the experiment, examine the three (or four) collection tubes and note
any color differences. Describe the contents of each tube in your laboratory journal.
24. Use a micropipettor to transfer the contents of each collection tube to a labeled
microcentrifuge tube. The label should include the identity of the sample (ex. HIC fraction
#1), your lab group initials or designation and the date. Make sure to use a fresh tip for each
sample.
25. Store your HIC fractions, along with the pre-purification supernatant sample, in the freezer.
Further analysis will be completed on these samples in the following lab activity.
26. Answer the remaining Conclusion questions.
Conclusion
1. Describe how proteins fold, mentioning at least three rules followed by the amino acids.
2. GFP contains a large number of hydrophobic amino acids. Describe how these amino
acids would be oriented in the protein.
3. How do you think the chemical properties of GFP can be used to isolate this protein from
others in a mixture?
4. By the end of the experiment, you should have a tube full of pure GFP. Describe one
technique you could use to determine if this protein has been isolated from the other
bacterial proteins or if additional proteins still remain in the mixture. (HINT: Think back to
another molecular technique you have used to separate and visualize items in a mixture.)
5. Explain how chromatography might be used in other career fields, such as forensic
science.
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Medical Interventions Activity 4.1.3 Protein Purification – Page 4