How Scientists Separate Proteins

Abstract

Have you ever had to sort a jumble of objects into piles, based on their type? Maybe laundry, or a big load of dishes, or while organizing the garage or a closet? Scientists have to do something similar when they want to study or isolate just a single type of protein. For example, a botanist might discover an exotic plant that is poisonous when eaten, but that also has great antibiotic properties. To help develop a new antibiotic for human use, he or she would have to separate the different plant proteins, and research which ones are toxic and which ones help fight off infections. One way scientists do this is by sorting the proteins, based on their size, using a technique called size-exclusion chromatography. In this science fair project, you can try your hand at this biochemical sorting technique and use it to determine the relative size of the green fluorescent protein (GFP).

Objective

To determine the relative size of green fluorescent protein (GFP), using size-exclusion chromatography.

Introduction

Manufacturing a life-saving drug, studying how the APOE protein contributes to Alzheimer's in humans, researching the effects of a bacterial toxin—these are all real-life instances where a biologist or a biochemist would need to separate a mixture of proteins. How are proteins separated? There are several methods, but one of the simplest ways is size-exclusion chromatography, which uses the size of a protein as a physical means of sorting. Different proteins come in different sizes, much like a grapefruit, an orange, and a lime are all citrus, but you can tell them apart by size alone.

In size-exclusion chromatography, a protein mixture, suspended in a special liquid, is poured into a column. The column, as shown in Figure 1 below, contains a suspension of tiny beads with different-sized macroscopic pores. As the proteins travel through the column, they get tangled in the beads. The larger proteins can fit in fewer pores and thus, have a more direct and quicker path through the beads. In contrast, the smaller proteins can fit through more pores and take a more meandering path as they get caught in various holes and take a longer time to flow through the column. As time passes, the special liquid, called an eluate, drips out of the column and into tubes. The initial drips of eluate contain the larger proteins, which passed through quickly, while the later drips contain the smaller proteins. Changing the collection tube under the column every couple of minutes results in multiple tubes of protein—each tube containing proteins of a different sizes.

Figure 1. Size-exclusion chromatography columns contain beads with various-sized pores. The larger proteins, which fit in fewer pores, take a more direct path through the column and elute first. The smaller proteins are slower to elute as they meander in and out of pores.

In this science fair project, you'll use size-exclusion chromatography to approximate the size of green fluorescent protein (GFP). GFP, first isolated from the jellyfish Aequorea Victoria, glows green when exposed to blue light. Because of its fluorescent properties, it is often used in cellular and molecular biology to track the expression of genes and the location of other proteins. In such tracking studies, it is important to keep the size of GFP in mind—if it is too large, it might not be able to move into some cellular areas, thus skewing the results of the study. To determine the size of GFP, you'll used a protein size-exclusion chromatography kit from Bio-Rad, which is explained in the Materials and Equipment section, below. The kit contains two other proteins: hemoglobin and vitamin B₁₂. You'll create a mixture of hemoglobin, vitamin B₁₂, and GFP, then sort them with the chromatography column. Since hemoglobin is reddish-brown in color and vitamin B₁₂ appears pink, you'll be able to tell which eluate samples contain each of these proteins. Using a UV light to make the GFP fluoresce, you'll determine which eluate samples contain GFP. From this information, you'll be able to determine the size of GFP, relative to the sizes of hemoglobin and vitamin B₁₂.

Terms, Concepts and Questions to Start Background Research

• Size-exclusion chromatography
• Eluate
• Green fluorescent protein (GFP)
• Hemoglobin
• Vitamin B₁₂
• Kilodalton

Questions

• How does size-exclusion chromatography work?
• What are the sizes, in kilodaltons, of hemoglobin and vitamin B₁₂?
• From where does GFP come?
• What other methods can be used to separate protein mixtures? What are their advantages and disadvantages?

Bibliography

• Tissue, B.M. (2000). Size-exclusion Chromatography (SEC). Retrieved July 28, 2009, from http://www.files.chem.vt.edu/chem-ed/sep/lc/size-exc.html
• Zimmer, M. (2009, June 21). GFP: Green Fluorescent Protein. Retrieved July 28, 2009, from http://www.conncoll.edu/ccacad/zimmer/GFP-ww/GFP-1.htm
• Wikipedia Contributors. (2009, July 24), Protein Purification. Wikipedia: The Free Encyclopedia. Retrieved July 28, 2009, from http://en.wikipedia.org/w/index.php?title=Protein_purification&oldid=303904910
• Wikipedia Contributors. (2009, July 28). Size-exclusion Chromatography. Wikipedia: The Free Encyclopedia. Retrieved July 28, 2009, from http://en.wikipedia.org/w/index.php?title=Size_exclusion_chromatography&oldid=304671149

Materials and Equipment

• Pipette, capable of dispensing 4-ml volumes
• Micropipettes, capable of dispensing 5- to 250-ul volumes
• Micropipette tips
• UV light
• Bio-Rad Size-exclusion Chromatography Kit from http://www.bio-rad.com
• Green fluorescent protein (GFP) sample
  1. You may be able to obtain this from a science teacher, or from a researcher at a local university. If not, you can use the Bio-Rad pGLO Bacterial Transformation from http://www.bio-rad.com and Green Fluorescent Protein Chromatography Kits from http://www.bio-rad.com to go through all the steps of creating a laboratory sample of GFP.
• Lab notebook

Experimental Procedure

Note: The Bio-Rad kit includes excellent detailed instructions, thus the procedure below is just an outline of the experiment. For step-by-step technical instructions, please consult the kit manuals.

Creating the Protein Mixture Sample

1. If you need to create your own GFP sample:
   1. Follow the instructions that came with the pGLO Bacterial Transformation kit to put the GFP gene into bacteria (i.e. transform the bacteria).
   2. Then use the Green Fluorescent Protein Chromatography Kit and the instructions contained therein to grow the GFP expressing bacteria, and purify the protein.
2. Once you have your GFP sample (either donated to you or created with the kits in step 1), combine 5 ul of the GFP protein sample with 10 ul of the hemoglobin/vitamin B₁₂ mixture from the Size-exclusion Chromatography Kit. This new 15-ul mixture will be your protein mixture sample for the size-exclusion chromatography experiment.

Performing the Size-exclusion Chromatography and Analyzing the Results

1. Carry out the size-exclusion chromatography, as instructed in the Bio-Rad kit's manual, using the GFP/ hemoglobin/vitamin B₁₂ mixture as your protein sample.
2. In the end, you should have 10 collection tubes from the columns. Note the color, in natural light, of each of these 10 tubes. Write your observations in your lab notebook.
3. Based on your visual inspection and background reading, which tubes contain hemoglobin? Which collection tubes contain vitamin B₁₂? Which tubes do you expect to contain GFP?
4. Look at all 10 collection tubes in a dark room, using only a UV light. Tubes containing GFP will glow green under the UV light. Record which tubes contained GFP and the relative brightness (which corresponds to the relative quantity of GFP) in each tube. Do your observations match your expectations?
5. Based on the results of the size-exclusion chromatography, what can you conclude about the size of GFP? Is it larger or smaller than hemoglobin? How about vitamin B₁₂? Can you make any guesses about its size, in kilodaltons?

Variations

• Size-exclusion chromatography can only help you determine a relative size for GFP. Devise a way to get a better estimate, in daltons, of the size of GFP.
• Try adding other proteins to the mixture and figuring out ways to isolate each one.
• How does pH affect size-exclusion chromatography? Devise an experiment to find out.