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Biology A/B

Identification of Animals by Serum Electrophoresis

Teacher Instructions

Index:

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:
    1. Agarose*
    2. Glycine*
    3. Tris Base*
    4. SDS*
  • Plastic tub for holding gel during stain/destain*
  • Masking Tape (if necessary)
  • Sheep, Rabbit, Cow, Pig, Moose sera standards, (can be self-prepped) prediluted with Running Buffer and stained with bromide blue.
  • Plastic Transfer Pipet
  • Plastic Wrap
  • 10 M NaOH*
  • Distilled water*
  • Coomassie Staining Solution*
  • Destaining Solution*
  • Plastic storage containers with lids for stain and destain
*These items may be shared among the lab groups in larger containers. Class Set
  • 1 Flat Bed Electrophoresis Apparatus (preferably 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 vertical 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.

Destaining Solution

(for coomassie destaining)

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

Special Instructions

Hand out lab instruction sheets one day before the Lab. Have the students read the handout and each one must write a prelab that consists of the Objectives, Materials and Equipment, Safety Precautions, Procedure, Hypothesis, and Data Table. This must be completed handwritten and in ink. The students should use the period they have received the handout to look up the iodine reagent in the Merck Index and record the safety precautions listed above. Instruct the students on the proper use of a pipet.

Before the lab, discuss the safety precautions necessary for running the high voltage electrophoresis apparatus. During the lab, present material discussing the effects of pH on amino acids, which amino acids are positive and which are negative at pH = 8.3, and how the charged field will move the proteins. Also discuss how the gel structure will work to separate the various proteins by size and shape in the electric field.

Method

(This is a sample of what should appear on the pre-labs. The days are inserted for convenience.)

Day 1

  1. Weigh out 0.010 grams of agarose for every milliliter of gel required. Place the agarose into a suitably sized test tube. (Example: for 25 ml of gel you would need to weigh out 0.250 g of agarose.)
  2. Add a volume of the tris/glycine running buffer for every milliliter of gel required. Add this to the agarose in the test tube. (Example: if you need to make 25 ml of gel you would need to measure out 25 ml of running buffer.)
  3. Heat the agarose in a boiling water bath until the agarose has dissolved into the buffer and the consistency is uniform.
  4. While the agarose is heating, pick up your unknown blood sample from your teacher. Use the centrifuge to separate the blood cells from the serum. Collect the serum into a 1.5 milliliter centrifuge tube by using a plastic transfer pipet.
  5. Cast the gel by pouring the hot agarose/buffer mixture into a cool gel casting tray (or casting platform if this is built into the electrophoresis apparatus, make sure the casting platform has well-taped ends). Insert the comb exactly 5.0 millimeters from the positive side of the electrophoresis apparatus. Teacher's note: While the gel solidifies is a good time to talk about how pH effects protein charge, how the charge to mass ratio effects the rate of motion of the protein through the gel, and how the gel physically separates different proteins by size and shape. temperature until tomorrow.
  6. Allow the gel to completely solidify, remove the comb, wrap the gel in plastic wrap and store at room temperature until tomorrow.

Day 2

  1. Remove the tape and plastic wrap and place gel in the electrophoresis chamber making sure that the wells made by the comb is on the positive side (it will already be there if your apparatus is a cast in place system, in this case remove the plastic wrap).
  2. Pour your tris/glycine pH 8.3 running buffer until the buffer submerges the gel and fills the wells.
  3. Load one of each of your standard sera and your unknown serum. Do not load the outside wells on the gel.
  4. Close the top to the electrophoresis chamber. Attach the chamber's electrodes to the power supply by connecting the positive side with a red jumper cable (do not use cables with alligator clips) to the positive outlet on the power supply and the negative side with a black jumper cable to the negative outlet on the power supply. If you reverse the polarity the proteins will run backwards. Correct connections can be verified by watching the opening minutes of the run.
  5. Check the top of the electrophoresis apparatus to make sure it is on snugly. Check all electrical connections and make sure that no metal is showing.
  6. Turn on the power supply. Set the power supply to put out constant voltage. Set the voltage to 100 volts. Set your timer for 30 minutes to check on the gel's progress.

    Preparation of running bufferto replace what is used.

  7. Weigh out 9.08 grams of Tris Base, 43.28 grams of Glycine, and 1.5 grams of SDS (Sodium Dodecyl Sulfate), into a 1 liter beaker and add 500 milliliters of distilled water.
  8. Add an appropriately sized stir bar and place the beaker onto a stir plate. Mix until dissolved.
  9. Stop the stir plate. Let the solution come to equilibrium. Test the pH of the buffer. If the pH is too low, add 10 M NaOH one drop at a time. After each drop, mix the solution and let the solution come to an equilibrium. If you have a pH meter you can monitor the pH continuously and when it approaches pH 8.3 stop the stir plate and let the buffer come to equilibrium before checking the pH. Continue this process until the pH of the buffer is at or very close to 8.3.

    Return to the running gel.
    (as the teacher you may have to stop the gel and put it into the staining solution and if you get the opportunity the destaining solution also.)

  10. When the bromophenol blue markers in the standard are within 5.0 millimeters of the end of the gel, turn off the power supply. Pour the buffer into a bottle of used buffer (this buffer can be reused). Mark your gel by poking a number of holes equal to your lab group, in the gel above (or in front of the wells) the wells. Do Not poke holes in the main area of the gel. Remove the gel from the apparatus and place it into a plastic storage container that is big enough for the gel to lie flat.
  11. Pour Coomassie Blue staining solution onto the gel. Let it sit for at least 1 hour (sitting longer does not hurt it).
  12. Clean up the electrophoresis equipment.

Day 3

  1. Return the staining solution to the bottle and rinse the gel with water.
  2. Pour destaining solution into the container with the stained gel. Let it come to equilibrium (the color of the gel will be the same color as the destaining solution).
  3. Pour destain that has come to equilibrium into the used destain solution container and later burn at an approved site. Add more destain to the gel, allow it to come to equilibrium and then pour the destain into an approved container for later disposal.
  4. Repeat step 19 until you have nice clean bands of serum protein in each of the standard lane and also in the unknown serum lane.
  5. Determine what animal your serum came from based on which serum standard your unknown matches.
  6. Slide gel unto a glass plate and allow it to slowly dry in the open air.

Day 4

  1. Clean up the lab.
  2. Obtain a photocopy of your dried gel.
  3. Turn in lab reports tomorrow.

Notes

The sera procedure is simple. Collect fresh animal blood, pig, sheep, cow, moose, caribou, etc. and centrifuge separating the sera from the whole cells. If the blood is not fresh the cells could undergo autolysis. Collect the sera in bullets (capped microfuge tubes - 1.5 ml) and freeze. It is handy to freeze the bullets in bullet racks, if not any plastic container with a lid works. Take one serum from each animal type. Make a dilution sera:running buffer of: 1:5, 1:10, 1:15, 1:20..... Record the dilution ratio for each animal type that gives a set of nice clean bands, no really concentrated bands, and the fine lines can still be seen. Run the marker band almost off the gel.

Also run a set of standards of each serum for your own information on a single gel, documenting the way the sera from each animal runs on your gels. This can be copied after staining/destaining if placed between two acetate sheets and you can enlarge the run using the copier. You may need to play with the contrast a little. There are subtle differences between the sera, but the students should be able to tell the difference (although there are some sera that are more distinctive than others). There is enough of a difference to identify an unknown serum when given the controls.

You can make it more interesting if it is part of a detective scenario. You have four or five suspects. One is a known pig farmer, another a dairy farmer, another a chicken farmer, another a moose hunter, etc. You have samples of fresh blood extracted from their clothing and from the crime scene. Identify the murderer.


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