Biology A/B

Animals by Serum Electrophoresis

Student Instructions

Index:

• Learning Objectives
• Materials and Equipment
• Safety Precautions - Toxic Waste Collection/Disposal Instructions
• Recipies
• Lab Procedure

Learning Objectives

1. Students will use a centrifuge to separate serum from cells and explain how density separates the two. (Physics)
2. Students will describe how the electrophoresis apparatus, with the buffer, works. (Chemistry/Physics)
3. Students will learn to properly cast agarose gels and prepare their own buffers. (Chemistry/Molecular Biology)
4. Students will describe the basic structure of proteins, the effect of pH on protein charge, and the need to carefully select the pH of the running buffer. (Biology/Molecular Biology)
5. Students will electrophorese varying concentrations of serum to discover the relationship between serum concentration and resolution possible by staining with coomassie blue. (Molecular Biology)
6. Students will run their unknown serum against a set of known serum standards and determine the animal source of their serum. (Molecular Biology)
7. Students will correctly write a lab report with the following sections: Objectives, Materials and Equipment, Safety Precautions (check reagents with the Merck Index or other safety reference), Procedure (stepwise), Hypothesis, Data Table, Observations, Conclusions, and Extensions (questions which point us where we want to go next or hypotheses as to why the lab did not run as expected). The underlined sections of the report will be finished before starting the lab. (General Science)

Materials and Equipment

• 1 set of the following for each 4 person lab group.
• Running Buffer (Tris/Glycine, pH 8.3)
• 1 unknown animal blood sample
• Distilled water
• Graduated Cylinder
• Electrophoresis Grade:
• Agarose*
• Glycine*
• Tris Base*
• SDS*
• Plastic tub for holding gel during stain/destain*

• Masking Tape (if necessary) Plastic storage containers with lids for stain
• Sheep, Rabbit, Cow, Pig, Moose sera standards, (can be self-prepped) prediluted with Running Buffer and stained with bromophenol blue.
• Plastic Transfer Pipet
• Plastic Wrap
• 10 M NaOH*
• Distilled water*
• Coomassie Staining Solution*
• Destaining Solution*

• 1 Flat Bed Electrophoresis Apparatus (preferrably a type that allows you to cast outside of the apparatus and then insert a ready gel when a lab group is ready to run otherwise it will take more time)
• 1 Power Supply
• Timer (to remind you to stop a running gel)
• Centrifuge
• 1.5 mL Microcentrifuge tubes (to store serum)
• 20 mL Pipettor
• 200 mL Pipettor
• 1000 mL Pipettor (nice to have but not absolutely necessary) or 1 mL serological pipet.
• Bio-Rad Prot/Elec pipet tips (these tips are small enough to load verticle gels and allow some
• student error when loading submersed gels)
• pH meter or pH test paper covering a range of pH 8-11
• Stir Plate with Stir Bar

Time Needed to Complete This Activity

This activity requires three to four 50 minute class periods to complete all phases. It can take less if the instructor or teacher assistants pre-make all of the reagents and gels.

Safety Precautions - Toxic Waste Collection/Disposal Instructions

Coomassie Blue stains all proteins. Use with gloves and aprons. Electrophoresis apparatus uses high voltage. Make sure the lid and all wires are securely in place before turning on the power supply. Destaining solution should be burned in a designated facility due to the presence of methyl alcohol.

Recipes

Agarose Gel

• 0.010 g Electrophoresis Grade Agarose for every milliliter of Electrode Buffer

Electrode Buffer

(for running gels, horizontal and vertical)

• 9.08 g Tris Base
• 43.28 g Glycine
• 1.5 g SDS

Procedure:

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.
• Adjust pH to 8.30 using 10 M NaOH.

Staining Solution

(for staining proteins, sera, enzymes, etc.)

• 250 ml MeOH
• 50 ml Glacial Acetic Acid
• 1 g Coomassie Blue
• 200 ml water

Procedure:

• mix all chemicals in proper order.
• Add 500 ml of water to dilute.

• 100 ml MeOH
• 50 ml Glacial Acetic Acid
• 350 ml water

Lab Procedure

On the first day pick up a complete set of lab group materials and equipment. You will make a gel today and let it set up for use tomorrow. Your instructor will tell you how many milliliters of gel you will be making. You will need to prepare a concentration of agarose that is 0.010 grams of agarose per milliliter. Weigh out the proper amount of agarose and add to it a volume of the running buffer that matches the volume of gel you need to produce.

Place your agarose/buffer mixture into a boiling water bath. You will notice that the agarose will melt into the buffer. When all of your agarose has melted and the consistency is uniform you are ready to cast the gel. If you have a casting tray that is separate from your electrophoresis chamber, make sure that the ends are completely sealed with tape. If your casting tray is part of your electrophoresis chamber, make sure that the plastic inserts for your casting chamber are securely in place. Mark your casting tray 5.0 millimeters from the negative electrode side.

Pour the gel into your gel casting tray. Use a plastic pipet to remove bubbles that form during pouring. Line up the comb with the marks made 5.0 millimeters from the negative electrode side, then insert your comb. Allow your gel to set up, this will take approximately 30 minutes. When your gel is set up remove the comb by pulling straight up on the comb. You have now created wells in your gel. Avoid removing the comb at an angle because it may tear the gel. Wrap your apparatus with plastic wrap to reduce loss of moisture from the gel.

The next day, unwrap your gel, remove either the masking tape or the plastic inserts from the electrophoresis chamber and if your casting tray is separate from the electrophoresis set your gel into the electrophoresis chamber. Pour running buffer into the electrophoresis chamber until the gel is covered by the buffer. Pour slowly so air is not trapped in the wells made by the comb. If air is trapped in the well, get another plastic transfer pipet and gently squirt buffer into the wells, dislodging the air bubbles.

Using clean pipets designed to deliver 10 ml, pipet 10 ml of each of your standard sera. Pipet your unknown into a lane near your standards. Your teacher may ask you to share your gel with one or more other groups so they may also pipet their unknowns onto your gel. Do not use the outside lanes on the gel. Your gel is now loaded and ready to run.

Put the cover securely onto the electrophoresis chamber and attach the leads to the power supply. The red lead goes to the red connection and the black lead goes to the black connection. Turn on the power supply and set it to deliver constant voltage. Set the voltage to 100 volts (if your power supply does not deliver 100 volts, set it to the voltage closest to 100 volts). For power supplies equipped with a start button, press the start button. As electricity flows through the chamber, you will notice that bubbles are forming at both electrodes. The positive electrode is attracting oxygen from the electrolysis of water. The negative electrode is attracting hydrogen from the same electrolysis. You will also notice that the dye in the standard sera will begin to move to the positive electrode. If it is going the other direction, check the connections and make sure that red and black leads and connections match.

Your gel will take most of the time available this class period. You have some time to replenish the electrode buffer solution. Using the preceding electrode buffer recipe, weigh out the proper amounts of tris base, glycine, and SDS (sodium dodecyl sulfate, a detergent). Add the proper amount of distilled water and mix this on the magnetic stirrer. As the solids dissolve, hook up the pH meter, set it to standby, and with the electrode arm immerse the tip of the pH electrode below the surface the pH 7.0, standard buffer. Check the pH, adjust the pH to read 7.0. Reset the pH meter to standby, lift the electrode out of the standard buffer and place an empty beaker under the electrode tip. Rinse the electrode tip with the distilled water in the squirt bottle. Wipe the electrode with a lab tissue (Kimwipe or other brand). Now you will immerse the tip of the electrode below the surface of the buffer dissolving on the magnetic stirrer. Check the pH. If the pH is below 8.3, add 10 M NaOH (sodium hydroxide) to the buffer one drop at a time. After the pH is above 8.1, stop the stirrer. Notice if the pH remains the same, drops, or increases above the reading that you just took. The amount of change is the amount of error caused by the motion of the ions in the buffer past your pH electrode. With this in mind continue to add NaOH dropwise to the buffer. Periodically stop the magnetic stirrer so you can get an accurate pH reading. When the pH equals 8.3 ± 0.05 you have successfully made the buffer.

Clean up your lab station and the buffer/pH station. Check the progress of your gel. If the blue dye of the standards is within 5 millimeters of the end of the gel, stop the electrophoresis by pushing the stop button on the power supply or turn off your power supply. Pour the used buffer into the used buffer bottle. Carefully mark your gel by poking a toothpick into the gel above the wells. If you are lab group 1, poke one hole. If you are lab group 2 poke two holes. DO NOT poke holes in the main area of the gel. Gently lift the gel out of the electrophoresis chamber and put it into a lidded plastic container that is large enough to hold your gel flat. If you are the first to finish, you will need to pour the coomassie blue staining solution onto your gel so that it is fully covered. If you are sharing a gel staining container, insert your gel and make sure there is enough staining solution to cover your gel. Let the gel sit in the staining solution for one hour or more. Clean up your electrophoresis equipment by rinsing it with cold tap water and then several times with distilled water. Let the apparatus air dry. If the class ends before your gel is done, inform your teacher who will shut down your lab and put the gel into the staining solution. You will still be responsible for pouring your buffer into the proper container and rinsing your equipment.

The next day pour off the coomassie blue stain solution back into its bottle. Place the stained gel into another lidded plastic container that is large enough to hold your gel flat. Pour destaining solution into the container until the destaining solution covers your gel. When the color of the destaining solution and the gel are the same color of blue, it has come to equilibrium and the gel will not destain any more. Pour off the destaining solution into the used destain container. Check your gel. If you have nice clean bands in the serum standards then you may stop destaining. If your gel is still too blue, continue the destaining process, making sure you allow the gel and the solution to come to equilibrium, until you get nice clean standards. Your used destain will be burned due to the presence of methyl alcohol (MeOH).

Compare your unknown serum with the standard sera. You should be able to determine the type of animal by matching the unknown with a standard that is the same. Place your gel onto a clear glass plate that is larger than the gel. Let it dry in the open air. It should be dry by tomorrow.

Obtain a photocopy of your dry gel. Then clean up the glass plate by softening the gel under warm running water. Slide the gel into the trash can (you may need to use your fingernails to scrape it off) and wash your glassware.

Observations:

Write down what you noticed with your five senses.

Conclusions

Use what you noticed, compare this with your hypothesis and textual material and explain what you think happened in the experiment.

Extensions

Explain ways to improve the lab, reduce discrepancies, reduce procedural errors, and things you can do to find out more about the blood sample.

Teacher Instructions

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