Materials Included In Kit

Additional Materials Required

Prelab Preparation

Preparation of 1X Tris-glycine-SDS running buffer and 1X Protein lysing buffer

1. Determine the amount of 1X solution necessary.
2. Multiply this number by 0.1 or 0.2. This is the amount of 10X or 5X concentrate needed.
3. Subtract the amount calculated in step 2 from the initial amount determined in step 1. This is the amount of deionized water that should be mixed with the buffer concentrate. Mix well.
4. Seal with Parafilm M or plastic wrap.
5. Label, cover, and store in a refrigerator.

   Note: Prepare enough Tris-glycine-SDS buffer solution to allow each group to cover the gel in the chamber to a depth of about 2 mm. Depending on the type of electrophoresis units being used, the amount of buffer needed could be as much as 300 mL per chamber. 90 mL of buffer is needed to make six agarose minigels. Make fresh buffer weekly.

Preparation of 1X protein destaining solution: Mix the 250 mL of protein destaining solution with 750 mL of deionized water.

Preparation of six 3% agarose minigels (6 cm x 6 cm gel)

1. Stir 3 g of agarose into 90 mL of the electrophoresis buffer in a 125-mL borosilicate Erlenmeyer flask. Stopper with a nonabsorbent cotton or foam plug.
2. Mark the height of the solution on the Erlenmeyer flask.
3. Dissolve the agarose by heating in a microwave, hot water bath or on a hot plate. Caution: Be careful not to superheat the solution because it will NOT boil until you disturb or disrupt it, whereupon it may spontaneously boil out.
   1. Microwave—30–40 seconds, stir, repeat.
   2. Hot water bath—do not boil the water.
   3. Hot plate—do not boil or scorch the agarose solution.
4. Heat until the solution is clear and agarose appears to be fully dissolved.
5. Stir frequently and do not allow solution to boil for more than a few seconds.
6. Use heat protective gloves to remove the flask.
7. Check the level of the solution. Add distilled water if needed.
8. To prevent damage to the casting trays, allow the agarose to cool to 55 °C before pouring.

Prepare the casting trays while waiting for the agarose to cool.

9. Attach the rubber dams to the ends of the casting tray or use tape to create the end walls.
10. Place the well-forming comb in the groove toward the end of the gel box.
11. Ensure the casting tray is on a level surface.
12. Slowly pour the melted agarose into the assembled casting tray being careful not to create bubbles in the gel. Use a stirring rod or pipet tip to push any bubbles to the edge of the casting tray. Only add enough agarose to equal the height of the indentations in the well-forming comb—do not fill the tray to the top.
13. Thoroughly rinse out the Erlenmeyer flask immediately.
14. Allow the gel to sit undisturbed for at least 30 minutes until the gel is firm to the touch. The set gel will appear opaque and somewhat white.
15. Once the gel is thoroughly set, carefully remove the well-forming comb by rocking it gently from side to side and then pulling it upward. Remove the end dams and carefully slip the gel out of the form.
16. Slide each gel into a separate resealable bag, add 5 mL of 1X Tris-glycine-SDS buffer, and refrigerate. Note: A solidified gel can be stored under buffer in a laboratory refrigerator for up to two weeks.

Safety Precautions

Electrical Hazard: Treat these units like any other electrical source—very carefully! Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Do not try to open the lid of the unit while the power is on. Exercise extreme caution in handling the protein staining solution—it will readily stain clothing and skin. The protein staining solution, protein destaining solution, and protein lysing buffer are mild skin and mucous membrane irritants. Wearing chemical splash goggles, gloves and an apron is strongly recommended. Wash hands thoroughly with soap and water before leaving the laboratory. Please consult current Safety Data Sheets for additional safety, handling and disposal information.

Disposal

Please consult your current Flinn Scientific Catalog/Reference Manual for general guidelines and specific procedures, and review all federal, state and local regulations that may apply, before proceeding. All solutions used in this lab may be disposed of down the drain using copious amounts of water according to Flinn Suggested Disposal Method #26b. Used gels may be disposed of in the regular trash according to Flinn Suggested Disposal Method #26a.

Lab Hints

• Gel preparation requires approximately 20–30 minutes plus at least an additional 60 minutes for the gel to solidify (overnight is better). Longer solidification times “harden” the gel, minimizing tears and creating more distinctive protein bands.
• When preparing agarose gels using a stirring hot plate, rotate a magnetic stir bar very slowly to diminish the number of bubbles in the agarose solution. 
• Enough materials are provided in this kit for 24 students working in groups of four or for 6 groups of students. This laboratory activity can reasonably be completed in three 50-minute class periods. The sample preparation will take most of one class period. The electrophoresis setup, sample transfer, electrophoresis, staining and destaining setup will take at least one 50-minute class period. Sample analysis will require part of an additional class period.
• A wide variety of tissues may be used for protein samples. We tested the above procedure on the following items—chicken, beef or fish as meat, heart, gizzard or liver; germinating bean, pea or corn seeds; albumin and lipase.
• The electrophoresis of samples will take 20 minutes to 2 hours depending on the voltage and equipment used to run the samples. Various protein tissue samples were tested using an Edvotek M-12 electrophoresis unit with two minigels at 125V for 30-45 minutes.
• Gel loading solution should not be diluted prior to addition to samples. Dilution is achieved when it is combined with the sample itself.
• Sucrose in the gel loading solution makes the sample denser than the Tris-glycine-SDS buffer causing the protein sample to sink into the sample well in the gel.
• Bromphenol blue in the gel loading solution migrates toward the positive electrode at the same rate as a medium sized protein. Xylene cyanole in the gel loading solution migrates at the same rate as a slightly larger sized protein.
• A 10 μL micropipet with disposable tips may be used instead of the disposable needle-tip pipets.

Teacher Tips

• Extend the lesson by incorporating a historical or new technology research project prior to the laboratory.
• Discuss how research laboratory training, techniques, and pricey equipment lead to a more refined result. This allows scientists to compare proteins between vastly different species.

Answers to Prelab Questions

1. Explain the function of each of following components of gel electrophoresis:
   1. Agarose gel—The agarose gel is the microscopic filter or sieve that separates large molecules from small molecules because of the size of the pores within the agarose.
   2. Wells in the gel—The wells hold the sample that will travel through the gel.
   3. Denaturing agent—The denaturing agent causes the protein molecule to lose its quaternary and tertiary structure creating a long rod-shaped molecule with a net negative charge.
   4. Electric current—The electric current provides the force that moves the charged samples through the gel.
2. List one important safety precaution that must be followed when performing any type of gel electrophoresis.

   For example, “Ensure that the work surface is dry, connecting wires and terminals are dry, do not open the electrophoresis chamber while the power is on.”

Sample Data

Observations

Answers to Questions

1. Scientists create samples using proteins with a known molecular weight (kDa) to help control for variability introduced during electrophoresis. The molecular weight of the unknown protein samples can be calculated by comparing the location of the unknown protein band to that of the known protein. In the figure below, calculate the molecular weight of the three unknown samples by comparing them to the protein marker in lane 1.
   {10929_Answers_Figure_5}
2. Slight changes in the DNA sequence cause changes in the resulting protein. Often times the protein change includes a change to the molecular weight in the protein. How can this fact be used to trace evolutionary changes in a protein?

   By comparing the protein banding pattern of similar species, slight differences in their protein composition can be visualized. These small changes can be compared to the protein banding pattern of a vastly different species to see which proteins remain similar even though the organisms are very different.

Introduction

Proteins are the workhorses of life. The functions of proteins in an organism are diverse—from structural support, to cell communication, as well as being able to function as an enzyme. There are thousands of different proteins within complex organisms, each with a specific structure and function. Every type of living organism has its own unique protein fingerprint.

Concepts

• Gel electrophoresis
• Protein structure

Background

Gel electrophoresis is a laboratory technique used to separate proteins, segments of DNA or RNA according to the size and the relative electric charge of the molecule. In 1950, the scientist Oliver Smithies (born 1925) determined that a gel made of starch acts like a molecular filter or sieve for proteins when it is positioned between positive and negative electrodes. Dr. Smithies discovered that proteins with different sizes, shapes and molecular charge move through the gel at different rates, with small charged fragments moving faster and farther through the maze of microscopic pores toward the electrode with the opposite charge (see Figure 1). For example, a negatively charged protein migrates through a gel toward the positive electrode which is called the anode. While Dr. Smithies’ original gels were very large, other scientists have refined his techniques leading to today’s small agarose minigels.

One hurdle scientists had to overcome was the complex shape and charge of many proteins. Large globular proteins found in complex organisms were not able to migrate through the pores of the gel. In addition, proteins with a no net charge would not migrate toward either electrode during electrophoresis. Denaturing agents act to unravel the complex quaternary and tertiary structures of the protein. Scientists found that when proteins are denatured they act like long rods with a net negative charge.

By altering the net charge of the proteins, scientists can load the protein samples into wells created on one side of an agarose gel. This creates a longer “run area” in which different sized proteins are able to separate. Protein samples are colorless as they run through the agarose gel so dyes are added to the samples before they are placed into the wells. The dyes act as a visual marker to show how far a small molecule and a larger molecule have migrated in the gel. Once the small molecule has reached a certain point on the gel, the electricity is turned off and the gel is removed to be stained to visually highlight the separate protein bands.

Brilliant blue is a nonspecific protein stain. A solution of brilliant blue is allowed to diffuse through the agarose gel where it binds to any proteins within the gel. The gel is transferred to a destaining solution in which unbound brilliant blue diffuses out of the gel leaving the bound stain attached to the unmoving proteins.

The types of samples that can be analyzed are endless. Samples of purified protein such as albumin or sucrase will provide distinct bands. Tissue samples such as meat or liver can contain hundreds of different proteins creating a protein streak within the gel. The protein streak from a germinating seed will be very different from a sample of fish muscle. Scientists can remove the streak of proteins using a special membrane and then test each area for specific proteins of interest, such as HIV proteins.

Experiment Overview

The purpose of this activity is to demonstrate the separation of proteins using the technique known as gel electrophoresis.

Materials

Prelab Questions

1. Explain the function of each of the following components of gel electrophoresis:
   1. Agarose gel
   2. Wells in the gel
   3. Denaturing agent
   4. Electric current
2. List one important safety precaution that must be followed when performing any type of gel electrophoresis.

Safety Precautions

Be sure all connecting wires, terminals and work surfaces are dry before using the electrophoresis units. Electrical Hazard: Treat these units like any other electrical source—very carefully! Do not try to open the lid of the unit while the power is on. Use heat-resistant gloves and eye protection when handling hot liquids. Protein staining solution will stain skin and clothing. Protein staining and destaining solutions are mild skin and mucous membrane irritants. Wear chemical splash goggles and chemical-resistant gloves and apron. Wash hands thoroughly with soap and water before leaving the laboratory.

Procedure

Part A. Tissue Preparation

1. Use a scalpel to cut a 1-cm² piece of protein tissue provided by your teacher and place into a mortar and pestle.
2. Using a graduated pipet, add 2 mL of protein lysing buffer into the mortar and pestle.
3. Grind the tissue sample in the mortar and pestle for 1 minute.
4. Allow the tissue samples to rest for 2 minutes, grinding occasionally.
5. Use a new, clean pipet to remove the liquid part of the sample into a clean, labeled microcentrifuge tube.
6. Clean the mortar and pestle with soap and water.
7. Repeat steps 1–6 with the five remaining protein samples.
8. Cap the six microcentrifuge tubes and place into the microcentrifuge. Centrifuge on high speed for 5 minutes.
9. Use a graduated pipet to transfer 0.5 mL of the liquid supernatant from one microcentrifuge tube to a clean, labeled microcentrifuge tube. If necessary, store at –20 °C (freezer). Repeat, using a clean graduated pipet for each of the remaining samples.
10. Use the dissecting needle to poke a hole in the top of each microcentrifuge tube. The hole will serve as a vent during the next step.
11. Place the microcentrifuge tubes into a floating microcentrifuge rack in a 95 °C water bath for 5 minutes.
12. If solids formed during step 11, transfer the liquid portion of the protein sample to a clean, labeled microcentrifuge tube.
13. Use a clean graduated pipet to add 5 drops of 6X gel loading solution to each protein sample. Cap the microcentrifuge tube and invert several times to mix the sample. If necessary store at –20 °C (freezer).

Part B. Loading a Gel

1. Assemble the electrophoresis unit according to the teacher’s instructions.
2. Place the electrophoresis unit in a horizontal position on top of a piece of white paper on a level table or countertop. Do not move the unit after loading the samples.
3. Gently place a gel into the casting tray with the wells toward the cathode (–) end of the unit.
4. Carefully position the gel and tray into the electrophoresis chamber. Caution: Be careful not to break or crack the gel. If the gel is damaged it should not be used as the breaks and cracks will affect the results.
5. Pour enough Tris-glycine-SDS running buffer into the unit to submerge the entire gel surface to a depth of 2–3 mm. If the gel begins to float, reposition it on the tray.
6. By convention, gels are read from left to right, with the wells located at the top of the gel. With the gel lined up in the electrophoresis chamber and the wells to the left, the contents of Sample 1 will be loaded into the well closest to you. Consequently, when the gel is turned so that the wells are at the top, “1” will be in the upper left corner.
7. Place the Protein Electrophoresis Worksheet on the counter in the same orientation as the electrophoresis unit. The small rectangles on the worksheet correspond to the wells in the gel.
8. Shake each sample microcentrifuge tube and lightly tap the bottom of each tube on the tabletop to mix the contents.
9. Withdraw 10–20 μL of Sample 1 from microcentrifuge tube by filling only the needle tip of a clean needle-tip pipet. Note: Fill the tip by squeezing the pipet just above the tip, not the bulb. Be careful not to draw the sample further up the pipet (see Figure 2).
   {10929_Procedure_Figure_2}
10. Dispense the sample into the first well by holding the pipet tip just inside the well. The sample will sink to the bottom of the well. Caution: Do not puncture the bottom or sides of the well. Do not draw liquid back into the pipette after dispensing the sample (see Figure 3).
    {10929_Procedure_Figure_3}
11. Record the sample name on the Protein Electrophoresis Worksheet in the appropriate well box.
12. Repeat steps 9 and 11 for the remaining samples. Use a clean needle-tip pipet for each sample. Load each sample into the adjacent well. Each student group will load 6 wells.

Part C. Running a Gel

1. Place the lid on the electrophoresis chamber and connect the unit to the power supply according to the teacher’s instructions.
2. Run the gel as directed by your teacher. Note: Bubbles should form along the electrodes in the chamber while the sample is running. The bubbles are the result of the electrolytic decomposition of water—hydrogen at the cathode and oxygen at the anode.
3. Turn off the apparatus to stop the sample migration in the gel when the first tracking dye is about halfway down the gel. (This may take 30 minutes to 2 hours.) The time necessary to run a gel depends on the type of electrophoresis apparatus and the applied voltage.
4. When the power is off, remove the cover and carefully remove the gel tray from the chamber. Place the gel tray on a clean paper towel. Note: Be careful not to break or crack the gel.

Part D. Staining the Gel

1. For best results, stain the gel immediately and destain overnight.
2. Wearing gloves, slide the gel off the tray and into the staining tray. Note: Do not stain the gel tray.
3. Gently pour about 40 mL of the protein staining solution onto the gel.
4. Allow the gel to stain for 2–5 minutes.
5. Pour off the stain into a glass beaker. The stain may be reused. Be careful not to damage the gel.
6. Remove the gel from the staining solution and wrap the gel in a paper towel.
7. Place the wrapped gel into the resealable bag.
8. Gently pour 150 mL of the protein destaining solution into the bag. Seal the bag and allow the gel to destain overnight.
9. Remove the gel from the bag and observe the gel bands. Place the gel on a piece of plastic wrap on a lightbox to see the bands more clearly.
10. Sketch the gel bands as they appear onto the Protein Electrophoresis Worksheet.
11. Consult your instructor for appropriate disposal procedures.

Student Worksheet PDF

10929_Student1.pdf