High School Science and Biotechnology:
Conceptual Development of Electrophoresis

Carole Bennett & Brian Hardcastle

Summer 1995

Introduction

Module Rationale

Teachers across the nation have indicated a readiness to participate in science education restructuring. For years now, the scientific community has asked the nation to reflect on the need for scientists and scientifically-literate citizens in our technology-driven world. The combination of changing requirements in our society and new insights about teaching and learning has brought a wide range of education stakeholders (educators, parents, and community and business leaders) to the realization that we must reform our educational system.

Education should foster students' natural curiosity, desire to learn, and allow them to explore and understand the complex interactions in the world around them. Knowledge of the concepts and methods of science is essential to prepare students for full participation in our diverse contemporary society. Scientific literacy is a national goal, as stated in Project 2061, Science For All Americans, and America 2000.

As outlined in 1992 by The National Science Teachers Association's text, The Content Core, the typical U.S. science program discourages real learning not only in its overemphasis on facts, but in its very structure which inhibits students from making valuable connections between facts. Most science programs in the U.S. secondary schools are organized in what is commonly called "a layer cake." Clearly, as the NSTA reports, the time has come for the educational edifice of facts and the layer cake to be dismantled. The emphasis on facts and rote learning and the difficulties students encounter in grasping theoretical considerations without a grounding in experience deters many from continuing in science.

Washington State University (WSU), with support from the National Science Foundation, has developed the Institute for Science and Mathematics Education through Engineering Experiences. The purpose of which is to invite cooperating secondary teachers into the field of engineering. By working with professors and graduate students at WSU teachers will acquire an understanding and appreciation of the field of engineering while formulating activities for the classroom that illustrate the connection between engineering and real- world application. This project is in concert with the current efforts to increase science and math literacy throughout the United States.

The module presented is a result of the work done in chemical engineering at Washington State University with Dr. Cornelius Ivory and Dr. Kwan Hee Kim. The project focused on biochemical separations using agarose and polyacrylamide gel electrophoresis. The main fields of application are biological and biochemical research, protein chemistry, pharmacology, forensic medicine, clinical investigations, veterinary science, food control as well as molecular biology.

Biotechnology

Biotechnology is an application of the data and techniques of engineering and technology for the study and solution of problems concerning living organisms. One area that is acquiring much acclaim is the identification of unknown proteins through various molecular properties. In most cases, molecules differ with respect to size, weight, configuration, or net electrical charge. Electrophoresis, gel filtration chromatography, and thin-layer chromatography (TLC) are techniques that work by taking advantage of subtle differences in these properties.

Electrophoresis

Electrophoresis is the process of measuring the migration of ions in an electric field. This is accomplished by placing a pair of electrodes in an aqueous solution of protein or amino acid. One type of electrophoretic separation is called Isoelectric focusing or IEF. Isoelectric focusing takes place in a pH gradient and can only be used for amphoteric substances such as peptides and proteins. The pH at which an amino acid has a net charge of zero is called the isoelectric point. This pH is given the symbol pI. In this cell, anions migrate toward the anode, and cations migrate toward the cathode. At its isoelectric point, an amino acid does not migrate toward either electrode in an electrophoresis cell. The isoelectric point of a given amino acid is a physical constant.

The type of electrophoretic separation that is demonstrated in the module is called Zone Electrophoresis. In this process a homogeneous buffer system is used over the whole separation time and range so as to ensure a constant pH value. The distance covered during a defined time limit are a measure of the electrophoretic mobility of the various substances.

An understanding of the acid-base behavior of amino acids, and also of proteins, is important for two reasons. First, it helps us to understand the solubility of these molecules as a function of pH. While amino acids are generally quite soluble in water, solubility is a minimum at the isoelectric point. To crystallize an amino acid or a protein, the pH of an aqueous solution is adjusted to the pI and the substance is precipitated, filtered, and collected. This process is known as isoelectric precipitation. Second, knowledge of isoelectric points enables us to predict the way components of mixtures of amino acids or proteins migrate in an electric field.

Electrophoretic separations can be carried out using a variety of mediums. In paper electrophoresis, a paper strip saturated with an aqueous buffer of predetermined pH serves as a bridge between the two electrode chambers. A sample of amino acid or protein is applied as a spot. When an electrical potential is applied to the electrode chambers, amino acid or protein molecules migrate toward the electrode carrying the opposite charge. Molecules having a high charge density move more rapidly than those with a low charge density. Any molecule already at the isoelectric point remains at the origin. After separation is complete, the strip is dried. In the case of paper electrophoresis, the paper is sprayed with a dye to make the separated components visible. The dye most commonly used for amino acids is ninhydrin.

Electrophoretic separations can also be done using starch, agar, certain plastics, and cellulose acetate as solid supports. This technique is extremely important in biochemical research, and is also an invaluable tool in the clinical chemistry laboratory. In 1949 Linus Pauling made a discovery that opened the way to an understanding of sickle-cell anemia at the molecular level. He observed that there is a significant difference between normal adult hemoglobin(Hb A) and sickle-cell hemoglobin(Hb S). At pH 6.9, Hb A has a net negative charge and Hb S has a net positive charge and on electrophoresis at this pH, Hb A moves toward the positive electrode and Hb S toward the negative electrode.

Sequencing of DNA, or reading the sequence of DNA bases along its length, can now be accomplished. In DNA sequencing, a radioactive label is added to single-stranded DNA. The DNA is divided into four groups that undergo different chemical treatments. The chemical treatments break the DNA into pieces that when separated reveal the positions of the bases on the original strand. The DNA pieces are separated by the utilization of electrophoretic techniques.

DNA fingerprinting is gaining much acclaim during the last three years. DNA fingerprinting takes advantage of the fact that large portions of the human genome are made up of repeated sequences of varying lengths that do not code for proteins.

DNA fingerprinting works by taking a small sample of human DNA that has been cut with a restriction enzyme. The resulting fragments are separated by size through the process of electrophoresis. The bands that result from using electrophoresis are then compared and measured against any other individual in the world.

Goals

General Goals

The first goal of this module is to provide students with a series of developmental activities that will give them experience in the separations of various solutions using electrophoresis. The second goal of the module is to provide students with practice in the application of information to a real- world setting, generation and analysis of data, synthesizing and developing an alternative protocol, as well as group and self evaluation of the research experience.

Student Learning Objectives

By the end of this module, the students should be able to:

1. Use accurately a micropipet and measure volumes in microliters.
2. Set up an apparatus which will separate a solution by electrophoresis.
3. Graph and predict the molecular weight of unknown proteins/amino acids or vice-verse using a graphing program.
4. Analyze protein/amino acid samples, based upon charge and migration, distance traveled and molecular weight using gel electrophoresis.
5. Compare and contrast the use of chromatography and electrophoresis as methods used in the separation of substances.

Brian Hardcastle
River Ridge High School
8929 Martin Way E.
Lacey, WA 98516
(360) 493-9604

Carole Bennett
Gaither High School
16200 N. Dale Mabry Hwy
Tampa, FL 33618
(813) 975-7340

Gel Electrophoresis

Abstract

Electrophoresis has applications in biology, biochemical research, protein chemistry, pharmacology, forensic medicine, clinical investigations, veterinary science and food quality control. Arne Tiselius received a Nobel Prize in 1948 for development of this moving boundary system technique for separation by electrophoresis in 1939.

Under the influence of an electric field, charged particles migrate to the opposite charged electrode. Because of varying charge and mass different molecules move at different speeds and are separated into fractions. Mobility is a characteristic parameter of each charged molecule and is dependent on the pK value of the charged group and its size. It is influenced by the type of molecule, concentration, pH of the solution, temperature and field strength as well as the nature of the support material during electrophoresis. Buffers are used to guarantee constant pH. The buffer ions are carried just like the other ions so the buffer concentrations are relatively large compared to the materials separated. i.e. Buffers may be 0.1M while only 10 ug protein is used. Because the relative mobility of each substance is dependent upon so many variables, the migration distance is compared to a standard which is used in the same experimental run.

Currently, gels are most commonly used as the support medium for electrophoresis although cellulose fibers or thin layers of silica have been used. They may be used in thin capillary tubes or as a film on glass or plastic plates. There are three major types of gel electrophoresis:

• Zone electrophoresis which uses a homogeneous buffer. Distance traveled depends entirely on the molecular mobility. This module demonstrates this medhcan which can separate dyes, proteins, or peptides, DNA.
• Isotachophoresis (ITP) used a discontinuous buffer system. The ionized sample migrates between a "fast" leading electrolyte and a "slow" terminating one. This method is used in quantitative analysis and will not be considered in this module.
• Isoelectric focusing which uses a pH gradient. This method is only used for amphoteric substances such as peptides and proteins. The molecules move toward electrodes until they reach a portion in the pH gradient where their net charge is zero. This is the isoelectric point and the electric field no longer influences the molecule. This method is most commonly used in qualitative analysis to separate mixtures of substances.

This module incorporates several experiments using zone gel electrophoresis.

• One utilizes a 1% agarose gel because of its simplicity of preparation and nontoxic characteristics. Agarose gels can separate molecules larger than 10nm. the pore size in these gels ranges from 150 nm at 1% to 500 nm at 0.16%. Pore size variation allows separation of a greater range of molecular size. Since agarose becomes cloudy in solutions > 1% it is usually used only in separation of high molecular weight proteins in research or commercial applications. This module introduces gel electrophoresis first with agarose gels and dyes because of its greater ease of preparation and then leads to more sophisticated material using polyacrylamide gels.
• Polyacrylamide gel separations developed by Raymond & Weintraub in 1959 allow greater control of pore size by varying both the cross linking agent between the acrylamide chains and by varying the ratios of catalyst. Larger amounts of acrylamide:cross linking agent produces smaller pore sizes and allows better separation of smaller molecules. Smaller pore size also produces sharper bands of molecules within the gel. Polyacrylamide gels have the advantage of using thin gels which allow faster separations, better defined bands, faster staining after separation and better staining efficiency. However, one possible consideration in use in schools is that acrylamide is listed as a neutotoxin and thus should be handled carefully by the instructor who makes the stock preparations.

REFERENCES:

Hames, B. D., & Rickwood, D. Editors. Gel Electrophoresis of Protein; A Practical Approach. 2nd Ed., IRL Press, NY, 1990.

Westermeier, Reimer, Electrophoresis in Practice, VCH Publishers, Inc., NY, 1993.

Schultz, B. & Hutchinson, N. Fred Hutchinson Cancer Research Center, SEP Dye Tchr WN, Sept., 1994.

Classroom Environment : High School chemistry, regular or advanced

Group size varies with activity as noted on each section.

Instructional Strategy

Introduce the unit using electrolysis of copper (II) chloride. Students should work in pairs or alone as this equipment is very inexpensive. The + copper ion moves to the cathode electrode and is visible. The - chloride ions travel to the anode and chlorine can be smelled. Have students problem solve how the ions could be slowed down (with a gel). Move into the concept of electrophoresis using gels. Elicit student predictions of which types of molecules would travel more rapidly.

Move into preparation of gel and electrophoresis using dyes. Students are then adept enough at the technique to work with polyacrylamide gels. The activity using the micropipet can be introduced before the gel activity with dyes or with proteins. Students do not have to use micropipets with the dye activity.

Time Frame:

Four 50-min class periods assuming prior practice with micropipets if activities terminate with dye electrophoresis

Day 1 - Introduce the concept of electrophoresis through electrolysis.

Day 2 - Demonstrate technique for pouring gel. Students prepare agarose solutions and pour gel onto labeled plates. (If time is limited, prepare agarose and keep warm in a hot water bath on a hot plate.)

Day 3 - Students set up gel boxes, add buffer, load the wells, run the electrophoresis and record results.

Day 4 - Students compare results and evaluate information. Discuss analysis questions generated by the experiment.

Day 5 - (Optional) Class simulation activity to use data to determine molecular weight of proteins

Schools continuing unit with activities using polyacrylamide gels and proteins or DNA refer to time frame in that module section.

Introduction to Movement of Ions in Solution, Electrolysis

Abstract

When an electric current is passed through a solution containing ions (charged atoms), electrolysis occurs. These ions travel to the electrodes and either lose electrons or gain electrons becoming neutral.

Materials

0.2 M CuCl₂, acetate sheet, electrolysis apparatus, 2 pencils, 9 volt battery, battery clip & wire with alligator clips on ends.

Protocol

1. Obtain materials and assemble electrolysis apparatus.
   1. Attach the battery clip to the battery.
   2. Connect the alligator clips to one end of each pencil so that the clip makes contact with the graphite point, not the wood.
2. Pour a small puddle (about 1- 1 1/2 inch) of copper (II) chloride on the acetate sheet.
3. Insert the pencil point electrodes into the solution as far apart as possible. Be sure the graphite points are in solution. (If the pencil point breaks, dry off and sharpen it.)
4. Observe any changes that occur on the cathode. (- charged, black wire) Look for reddish, brown deposits on one electrode.
5. Observe any changes occurring near the anode. (+ charged, red wire) CAREFULLY smell the gas released by wafting the vapors toward your nose and inhale carefully.
6. As soon as you make observations, stop the electrolysis by removing the electrodes from the liquid.
7. Rinse the electrodes and wipe clean before returning to the stock area.
8. Rinse the copper chloride solution down drain and dry the acetate.

Analysis Question

1. What substance is the reddish, brown deposit on the cathode?
   
2. Why did the Cu²⁺ ion travel to this electrode?
   
3. What substance was released at the anode? Which ion traveled to this electrode?
   

Extension

1. Protein molecules become charged in acidic or basic solutions. If a protein becomes + charged, which electrode would it move toward? What if it becomes - charges instead?
   
   
2. Ions such as Cu²⁺ and Cl^1- travel very rapidly toward electrodes. How would the speed of proteins with molecular mass in the thousands compare with the copper (II) ion and the chloride ion?
   
   
3. Scientists can separate proteins using a similar method called electrophoresis because the heavier molecules move more slowly and travel a shorter distance in a given time. A method called "gel" electrophoresis uses a gel matrix of long ,tangled molecules. This makes it even more difficult for the larger molecules to travel and aids separation. Your next activity will be gel electrophoresis. Look in a reference and see how DNA molecules are separated.
   
   

MICROPIPET PRACTICE ACTIVITY

Abstract

Most separations of proteins are on a very small scale and use micro amounts of liquids. This requires you to manipulate a hand-held instrument called micropipet. This activity will familiarize you with their use and gives a quick way to illustrate if you are using them properly.

Materials:

Kit with foam block holding plastic microfuge tubes of red, green, blue and yellow food dyes, 4 empty microfuge tubes, pipet tips, filter paper, micropipet

Protocol:

1. Use of micropipet
   1. Volume setting
      1. Look at micropipet to determine the volume range, 0.5 uL - 10 uL or 2 uL- 20 uL
      2. Find out from your instructor if this pipet has a volume lock setting. If so, release the lock as shown.
      3. Turn the control knob to select the needed volume. DOUBLE CHECK IT.
   2. Attach a pipet tip.
   3. Handle the pipet with thumb on the release button and in a vertical position. NEVER PIPET LIQUID WITHOUT ATTACHING A TIP TO THE PIPET. NEVER LAY PIPET DOWN WITH LIQUID IN THE TIP.
   4. Filling:
      1. Press the control button down to the first stop.
      2. Immerse the pipette tip 2-mm into the liquid.
      3. Allow the control button to glide back slowly.
      4. Slide the tip out along the inside of the container.
      5. Wipe off any external droplets on the tip with lint-free tissue.
   5. Dispensing
      1. Immense tip into the liquid.
      2. Slowly press the control button completely to activate the blow-out feature.
   6. Eject the tip.
2. Practice using the pipet with samples of water to become comfortable with the feel of the control button stops.
3. Formation of four new colors:
   1. Place a 3 uL sample of the basic dye colors (red, green, blue & yellow) on filter paper and label spots with pencil.
   2. Use the chart below to mix the required mL of basic colors in correct clean, empty microtube.

      	Red	Green	Blue	Yellow
      Teal		5 uL	15 uL
      Rose	15 uL		5 uL
      Orange	6 uL			14 uL
      Chartreuse		2 uL		18 uL

   3. Mix all dyes into a single droplet in the bottom of each microtube. (Use vortex mixer or clean, plastic toothpick)
   4. Place a 3 uL sample of the mixed colors on the same piece of filter paper that you placed the basic colors.
4. Clean up.
   1. Wash the microfuge tube with the mixed colors (teal, rose, orange & chartreuse) with distilled water.
   2. Replace the clean tubes in kit for next group.
   3. Clean up area.
   4. Replace micropipet as indicated. by your instructor.
5. Attach the filter paper to a sheet of paper with your name and date for grading.

Analysis Questions:

1. Compare the sizes of dye spots on your filter paper. The sizes of the spots should be identical if your technique is correct.
2. Compare the colors produced for teal, rose, orange and chartreuse with a standard. This also indicates your ability to follow directions and handle the micropipet correctly.

How to Make a Gel Plate

Abstract

Electrophoresis involves charged molecules moving within a gel matrix of tangled molecules. In this case the gel is agarose, a derivative from seaweed. Prepare your gel by measuring a quantity of solid and adding it to a buffer solution. After heating to dissolve the solid you need to pour the hot liquid onto a glass plate and then form small slots in the gel called wells. The gel will harden and can be used tomorrow.

Materials

Centigram balance, weigh boats or paper, agarose powder, 1X TAE buffer, graduate, microwave or hot plate, hot gloves, glass stirring rod, 125 mL beaker or flask, goggles, 5" x 5" glass plate, comb to form wells, small amount of buffer

Protocol

1. Add 0.50 g solid agarose powder to 50 mL buffer in beaker or flask.
2. Heat until all particles are dissolved --- about 30 sec to 1 minute after solution boils to remove all gas. (Use a hot plate or microwave.) If using a microwave heat for 45 sec first, then heat in increments of 10 sec to prevent liquid boiling over.
3. Keep flask in a 60^o C water bath until ready for use.
4. Label underside of glass plate for identification. Place glass plate on flat surface, label side down.
5. Using a glass stirring rod as a guide, carefully pour hot agarose solution down rod so that the gel is positioned on the outer edges first. Fill in the center last. The gel at the edge cools first and surface tension allows more gel in the middle. If you aren't careful the gel will run off the glass. If you have an accident, allow to cool slightly and scrape off. Place gel back in beaker and re-heat.
6. Immediately place a comb in the gel about mid-way from one end. Use the diagram on the dye separation sheet as a guide.
7. Let the gel harden 10 min. Add a few mL of buffer on surface of hardened gel and remove comb gently from one side first and keep comb on a slant.
8. Store in a container or Ziploc bag for use tomorrow as directed.

Separation of Dye Molecules using Gel Electrophoresis

Abstract:

In this activity several biological dyes will be placed in gel wells and separated using gel electrophoresis. The dyes are charged and will move toward either the cathode or anode at different speeds. Thus a mixture of dyes can be identified if compared with dye patterns of individual dyes.

Materials: for a group of 4

Protocol

1. Set up electrophoresis apparatus near the power supply as shown by instructor.
   1. Measure the pH of the TAE buffer
   2. Add 1X TAE buffer to the buffer wells
2. Obtain your prepared gel. Carefully slide it out of the Ziploc bag and place in its position in the electrophoresis apparatus as suggested by your instructor.
3. Obtain sample dyes
   1. Obtain sample dyes for your group by pipetting 20 uL of each dye from the class supply into the appropriately labeled microtube. This is 15 uL to load in the well plus a little extra.
   2. Using a micropipet with special loading tip or micro Beral pipet, add small samples (about 15 uL) to each slot in the gel.
   3. In your data table, carefully record which dye is in which slot.
   4. If you have a commercial electrophoresis apparatus, go to step 5.
      If you have a non-commercial apparatus, do the following:
      1. Place two thickness of paper towels so that one end is draped on the edge of the gel and the other is immersed in the buffer well. This will make a electrical connection between them. Repeat on the other side of the gel. See diagram below
      2. Double check your buffer solution to be sure that there is very little distance (less then 2") between the top of the solution surface and the gel plate. If needed, add more solution.

      

   5. When all samples are loaded, close the lid on the gel box or use the Plexiglas plate provided as a cover.
   6. Student groups will share power supplies. Connect the electrodes to the gel box and the power supply connecting red to + and black to -. Turn on power supply and set it to 100 voluts. Check the milliamp output. Electrophorese for 10 min total.
   7. Observe and record changes in dye position in the gel after 5 minutes on your data sheet.
   8. After about 10 min, turn off the power. Unplug the electrodes and open the gel box.
      1. Measure the pH of the buffer at each end of the box.
      2. Lift out the gel deck and gel. Place them on the lab table on the plastic wrap, wiping off excess buffer.
      3. Record your observations on your data table.
   9. Make a permanent record of your gel by performing ONE of the following.
      1. Place a piece of acetate sheet on top of the gel. Using a permanent marker pen, mark the wells and each dye spot. Note the + and - ends.
      2. Set a piece of blot paper on top of the gel and press straight down for about 5 sec. Dye will transfer from the gel to the blot paper.
   10. Place your gel in plastic Ziploc bag to prevent drying.
   11. Measure the distance that each dye sample traveled on the gel. You can use the acetate sheet or gel for this. Measure in mm from the lower edge of the well to the center of the dye spot. Record data.
   12. Replace equipment as directed. Do not discard buffer. Place in container labeled "Used buffer" for reuse next period.

Gel Electrophoresis Dye Lab Report Sheet

Name ________________________
Record all data neatly, in ink.

Analysis questions

1. Record observations of the gel after 5 minutes of electrophoresis.
2. Draw an accurate illustration of your gel results on the back.
3. Toward which electrode did the greater number of samples run?
4. Compare your food dye results with another group. Was the distance traveled the same for different brands of red food dye? Propose a reason.
5. Which dyes seemed to be composed of more than one pigment?
6. If your group ran a mixture, what dyes were in the sample? Give evidence to support your answer.
7. What molecular properties were used by gel electrophoresis to separate molecules?
8. If you were asked to improve the separation of these dyes, what are some of the variables you could modify in your experiment?

Determination of Molecular Weight of an Unknown Protein

Abstract

Proteins move through gels during electrophoresis at different rates because they have different molecular weights. It has been established that as proteins move through gels, the distance traveled by a given protein is proportional to the log₁₀ of the molecular weight. It is a linear relationship, but the heavier proteins travel a much shorter distance.

Every time a scientist performs gel electrophoresis on proteins he/she uses a standard mixture which is called a "marker". The molecular weights of the proteins in this mixture are known and can be used to determine weights for unknown samples. When given the data for proteins of known weight in a standard, you will be able to determine the molecular weight of an unknown after you graph the data and use them to make interpolations.

Materials:

graph paper, straight edge, calculator

Protocol

1. Determine the log₁₀ of the molecular weight of the proteins that are in the standard mixture.
2. Graph the data neatly on graph paper.
   1. The log₁₀ of the molecular weight is traditionally graphed on the Y axis and the distance traveled on the X axis.
   2. Plan the increments on each axis to distribute the variables on the graph as much as possible.
   3. When the points are placed properly, join them with the "best fit" straight line you can give with these data. The line should go through the origin.
3. Using this line, determine the log₁₀ of the molecular weight of the protein given the distance traveled.
4. Calculate the molecular weight of each protein by finding the antilog of these value.
5. Look up the correct molecular weights and determine your percent error.

Separation of Proteins
Using Polyacrylamide Gel
Electrophoresis

Abstract:

Polyacrylamide gels were first used for electrophoresis in 1959. They are chemically inert and mechanically stable. By chemical co-polymerization of acrylamide monomers with a cross-linking reagent a clear transparent gel exhibiting very little electro-osmosis is obtained. The pore size can be exactly and reproducibly controlled by the total acrylamide concentration and the degree of cross-linking.

The protein samples are run through a stacking gel prior to entering the running gel phase. In the stacking gel the protein concentrates because the solvent is made discontinuous. This concentration step enhances the final resolution obtained.

Students will be assigned to make a gel of a particular pore size based upon the percentage of polyacrylamide utilized (5%-15%). A marker protein Kaleidoscope will be used as a comparison known to unknown proteins. Students will graph the log of the molecular weights of the proteins vs. the distance traveled (cm.) in the gel. From this information students will determine, based on their graphical information, the molecular weights of the proteins of samples.

After given the known values for these proteins, students will calculate their percent error and determine viable sources of error.

As an extension to this lab, students will be asked to develop a gradient gel, 5%-15%, in order to generate a more complete separation.

Instructional Strategy:

In this exercise students will generate protein separations utilizing different pore sizes. These gels will be posted so each student can view the percent of polyacrylamide vs. the quality and quantity of the separation. Students will then be asked to infer conclusions from this data. This format lends itself to a guided inductive inquiry process. By using inductive inquiry the processes of observation, inference, classification, formulating hypotheses, and predicting are all sharpened or reinforced by the experiences.

After completing part one of the lab experience, students will be asked to devise a system that will result in a gel gradient. The purpose of which is to decrease, progressively, the pore size to "trap" more proteins throughout the separation. The process developed, by the students, will be analyzed for effectiveness based upon the separation of low and high molecular weight proteins. This problem solving technique implies a certain degree of freedom to explore the problem and to arrive at a possible solution.

Stock Solutions:

SOLUTION A--Acrylamide-BIS, 30:0.8; 300 grams acrylamide + 8 grams N'-N'-bis methylene-acrylamide. Make up to 1000 mL with water; Acrylamide from Biorad.
SOLUTION B--181.5 grams Tris (Biorad) + 500 mL water. Adjust pH to 8.8 with HCl. Make up to 1 Liter with water (1.5M).
SOLUTION C--10% SDS in water. SDS from Biorad.
SOLUTION D--60 gram Tris + 400 mL water. Adjust pH to about 6.8 with HCl. Make up to 1 Liter with water (0.5M).
SOLUTION E--10% Ammonium persulfate (AP) made fresh daily.

Caution!!! ACRYLAMIDE IS A POTENT NEUROTOXIN. Avoid breathing acrylamide dust. Skin contact with acrylamide solutions and mouth pipetting of acrylamide solution should be strictly avoided!

Electrophoresis involves the application of potentially dangerous voltages to the gel and gel reservoirs. At no time should any part of the electrophoresis set-up be touched following application of current to the apparatus.

Protocol:

Step 1: Determining the Gel Time

1. The gel time for the running buffer and stacking gel should be between 15-30 minutes. In order for this to occur a ratio between the polyacrylamide gel and the initiator (TEMED) and cross linker (AP) must be determined. It is recommended that the instructor pre-determine the appropriate ratios. (HINT: In 40 mL of gel we added 50 uL AP & 10 uL TEMED)

Step 2: Running Gel Stock

1. The following is a general formula for making a 15% Gel. All calculations are based on these percentages with a resulting volume of 40 mL.
   Running Gel 15%
   Solution A-- 20 mL
   Solution B-- 10 mL
   Solution C-- .4 mL
   add water to 40 mL
   

Step 3: Sealing the Gel Box(the following instructions are for a vertical gel box)(see "Electrophoresis of Dyes" representing an example of a horizontal run)

1. Assemble the gel box with spacers and clips.
2. The amount of gel needed to seal the box depends upon the dimensions of the unit. As a general guide the following applies. Extract 10 mL. of the Running Gel Stock and dispense into a 50 mL flask.
3. Add the cross linker and initiator in appropriate ratios. (To expedite the sealing process an increase of 20% initiator can be utilized)
4. The best way to seal the box is to incline the gel box and pour the liquid sealing gel at the base. One can also tape the base of the box and then fill through the space between the plates. Caution!! There is a possibility of pulling the gel out when removing the tape. You probably could leave it and everything will be fine.
5. After the bottom of the box has solidified a small layer of sodium dodecylsulfate(SDS) may remain on top of the gel seal. Simply pour off the excess SDS.
6. Place box on its side and seal following the same process in b and c. This time inject the gel seal between the glass plates. About .2 cm above the spacer should be enough.

Step 4: Running Gel

1. Extract the remaining 10 mL of Running Gel Stock and place in a 50 mL flask.
2. Add pre-determined amounts of initiator and catalyst, mix and dispense. Fill up to a point so as the proteins will migrate through 1 cm of stacking gel once the comb is inserted. Let solidify.

Step 5: Stacking Gel

1. The following is a formula for preparing the stacking gel. Pour the stacking gel just before use (to maintain the pH gradient between the stacking and running gels): remember to pour off the SDS overlay before pouring the stacking gel!

Stacking Gel
Solution A-- 1.3 mL
Solution D-- 2.5 mL
Solution C-- 0.1 mL
Water-- 6.0 mL
AP-- 0.1 mL
TEMED-- 10 uL

2. Extract and dispense the above recipe for the stacking gel in a 50 mL flask. Mix and pipette the stacking gel filling the total volume of the space provided between the glass plates.
3. Insert the comb let solidify.

Step 6: Preparing the Protein Samples(See teachers guide for sample recommendations)

1. Place 5mg of each protein into a small microtube.
2. Add 3.3 mL of water. This will be you Stock Solution for each protein!
3. For each protein place 10uL Stock Solution + 10uL of water + 20uL(2x)GSB + 5 uL B-mercaptoethanol in a microtube.
4. Place microtubes from '3' in boiling water for 2 minutes.

   2x GSB-Gel Sample Buffer
   Solution D-----------------------2.5 mL
   Solution C-----------------------2.0 mL
   Glycerin-------------------------2.0 mL
   0.1% Bromophenol Blue---------1.0 mL
   Water---------------------------2.3 mL
   Dilute the sample, preferably in water, 1:1 with 2x GSB. Add beta-mercaptoethanol to 10% final concentration.

Step 7: Loading Samples and Running the Gel

1. Remove the comb.
2. Fill the wells with Running Buffer. At the same time fill the upper and lower reservoirs with Running Buffer.
3. Dispense 15 uL of each sample into a well. Make sure to replace the pipette tip after each load. Fill the well from the bottom up! *Remember to use a marker to be used a method of comparison. Kaleidoscope(Biorad) is a good marker. (See chart listing band color vs. molecular wts.) Kaleidoscope

   Protein Band	Color of Band	Molecular Weight
   Myosin	Blue	201,000
   B-galactosidase	Magenta	134,000
   Bovine Serum Albumin	Green	81,000
   Carbonic Anhydrase	Violet	41,500
   Soybean Trypsin Inhibitor	Orange	31,800
   Lysozyme	Red	17,900
   Aprotinin	Blue	7,700

4. Connect cooling lines if available.
5. Attach electrodes and run the gel at 60 Volts (approx. 40milliamps) through the stacking gel.
6. As soon as the samples start flowing into the running gel increase the voltage to 150 Volts.
7. Run the gel until the samples are within 2 mm of the seal at the bottom of the box. Turn off the power. Disconnect the electrodes. (Turn off cooling lines as well, if available)

The following steps involve the preparation of the gel for data analysis. It is recommended that gloves be worn at all times.

Step 8: Fixing the Gel

1. The following is a recipe for making the fixing solution.

   Fixing Solution
   200 mL Ethanol
   40 mL Acetic Acid
   200 mL Water
   

2. Place gel in a Pyrex dish.
3. Pour fixing solution over gel.
4. The gel should be in the fixing solution for 30 minutes.

Step 9: Gel Staining Method(Using Coomassie Blue)

1. The following is a recipe for making the staining solution.

   Staining Solution
   1.1g Coomassie Blue (Biorad)
   200 mL Methanol
   40 mL HOAc
   200 mL Water
   (Vacuum Filter before use)
   This solution may be used 5x before discarding
   

2. Place the gel in a Pyrex plate. Add the staining solution until the gel is covered.
3. Agitate occasionally. Staining time 30 minutes.

Step 10: Destaining the Gel

1. The following is a recipe for making the destaining solution.

   Destaining Solution
   7.5% Acetic acid
   10% Ethanol
   

2. Remove the stain from the dish, taking care not to break the gel.
3. Wash the gel once in de-ionized water.
4. Destain by adding to the dish 300 mL of Destaining Solution.
5. Upon addition of the destaining solution, the protein bands should be immediately visible. This should improve over time.
6. Destain 2x 30 minutes.

Step 11: Photographing Gel

1. Place the gel on a piece of Saran wrap. Use a white background to aid in viewing the protein bands.
2. Place a ruler (cm scale) next to the sample and photograph.
3. If a camera is not available have students use graph paper to make a depiction of the results. (Make sure they keep track of the scale)
4. Discard Saran wrapped gels in waste basket when completed.

Students are to plot the relative mobility as compared to bromophenol blue (RBPB) for each of the marked proteins. The data are generated from the start of the running gel. Students will plot the RBPB against the log of the molecular weight for each protein marker. The molecular weight of an unknown protein bands can then be determined. Once determined the students will calculate the percent error for known values of the protein bands. Students will then follow through with the standard procedure for lab presentation as defined by the following: (The students should be with in 10%-15% of the known values for their calculated molecular weights)

Questions

1. What is the advantage of polyacrylamide gel electrophoresis over using agarose gels? List two factors.
2. How does the polyacrylamide percentage of your gel compare with that of others using the same and different percentages (In terms of quality and quantity of protein separation)?
3. What appeared to be the best percentage of polyacrylamide utilized during the separation?
4. Design an experiment that would exhibit a greater range of separation. Be prepared to state your hypothesis.

Conclusions

• Summary of Lab Experience: highlight electrophoretic technique utilized
• Explanation of Observations: Based upon your research and general scientific knowledge explain what listed in your qualitative summary.
• Sources of Error: List three possible sources of error in your lab and how these would effect the outcome.

Data Analysis and Extension

After class discussion of results, the concept of using a linear gradient will be either dynamically generated or funneled by the teacher. Students will be given two 10 cc syringes, various tubing, clamps etc., needed to set-up a linear gradient gel. The students will also be given an article take from Electrophoresis in Practice by Reiner Westermeier (1993) summarizing the effect of using a linear gradient.

After testing their system, groups will poster their results. A group presentation will follow where each group will explain their system and results. Each member of the group will be assigned a particular section of the presentation: (1) Discussion of design; (2) Presentation of data; (3) Comparison of data to prior nonlinear gradients; and (4) Sources of error, plus the highlighting the areas of improvement in the design.

Teacher Guide
for
Separation of Proteins
Using Polyacrylamide Gel
Electrophoresis

Answers to Text Questions:

1. (a) It is easier to regulate the pore size consistently with polacrylamide gels, (b) The best application of gradient gels is with polyacrylamide gels. *Students may have a variety of answers pertaining to their lab sequence as well.
2. Students will notice that the optimum percentage of polyacrylamide gel is dependent upon the molecular weights of the proteins used.
3. Data dependent
4. Student generated

Teacher Notes

The electrophoretic technique used here employs polyacrylamide gels. This technique exploits differences in molecular size and charge for the purposes of separation. The gel components are not charged and can be varied in a known manner to produce gels of various specific pore sizes. An effective pore radius of 0.5-3.0 nm can be obtained by adjusting the total acrylamide concentration and the concentration of cross-linking reagent in the polymerization mixture. Crosslinking agents other than BIS/acrylamide have also been used that have the advantage that they can be incorporated into the gel and later have their crosslinking bonds readily broken. Thus, it is possible to redissolve the portion of the gel containing a macromolecule of interest.

Gelation takes place more slowly at lower pH values because the free form of the base is required to catalyze the reaction. The polymerization rate is highly temperature dependent, hence the temperature must be kept constant.

Electrophoresis of Proteins
Since proteins are amphoteric, the pH of the electrophoresis system must be chosen bearing in mind the isoelectric points of the proteins which are to be separated.

The electrophoresis system in this experiment employs a detergent, sodium dodecyl sulfate (SDS). The SDS system involves concentrating protein samples into very thin layers using a discontinuous voltage gradient and then electrophoresed onto a column of polyacrylamide gel. The concentration step enhances the final resolution obtained.

Principles of Electrophoresis
The protein concentrates because the solvent is made discontinuous. Chloride and glycine solutions are electrophoretically in series. The movement of ions (the current) must be the same throughout the system. When a voltage is applied the glycine and chloride will move toward the positive electrode. This concentrating step is called "stacking" and occurs in the upper gel.

A tracking dye is added to the protein or the upper buffer before electrophoresis. It has a mobility which is dependent on the pH in this region and is intermediate between chlorine and glycine. It is customary to indicate the relative mobility of substances on the gel as a ratio of the distance traveled by the substance to that traveled by the tracking dye.

References: Freidfelder, pp 211-234
Original Literature: Shapiro, Vinnela & Maizel
References: Comm. 28, 815 (1967)

Electrophoresis of DNA Using Agarose Gel
(Understanding the Role of Restriction Enzymes)

Abstract:

Agarose electrophoresis is the standard method for separation, identification and purification of DNA and RNA fragments. Horizontal gels are used for these nucleic acid separations: the agarose gel lies directly in the buffer. This prevents the gel from drying out. The gels are stained with ethidium bromide and the bands are visible under UV light.

Using proteins called restriction enzymes, genes can be cut at specific DNA sequences. More than 75 different kinds of restriction enzymes are known, and each one "recognizes" and cuts DNA at a particular sequence. The accuracy of these enzymes is amazing. They will not cut any sequence other than the one they recognize, even if five out of six base pairs are identical to their recognition site. Restriction enzymes make it possible to cut DNA into fragments that can be isolated, separated, and analyzed.

Instructional Strategy:

Cooperative learning increases achievement, stimulates cognitive development, promotes active learning, increases self-esteem, and enhances positive attitudes toward school. Increasingly, business and industry require that people work together in production teams or in problem-solving teams. Therefore, learning to work effectively in a group is important; cooperative problem-solving groups in the chemistry lab help students build and hone their skills.

Students will work in lab groups of two. Once completed they will coordinate their presentation of data, as described in the conclusions, with another team. Students presentations will be graded based upon the Graduation Project Scoring Rubric utilized at River Ridge High School, Lacey, Washington.

*Companies sell pre-cut DNA from an assortment of restriction enzymes. These may be purchased to save preparation time!

Protocol:

Step 1: LB Plates and Broth

1. Mix in a 1000 mL flask (does not need to be dissolved) the following substances; 5g Tryptone, 2.5g Yeast Extract, 5g NaCl, 7.5g Agar, 500 mL water.
2. Autoclave on slow exhaust for 30 minutes.
3. Cool for 15 minutes.
4. Add 500 microliters of ampicillin.
5. Pour the plates.
6. Flame out the bubbles.
7. After the plates have set, dry the lids in the tissue culture room with a blower and germicidal lamp on.
8. To make the LB broth exclude agar and autoclave everything 30 minutes on slow exhaust.
9. Pour into 100 mL vials and autoclave again.

Step 2: Growing Cultures

1. Add 100 microliters of ampicillin to 100 mL of LB.
2. Pipette 5 mL LB into each culture tube and inoculate.
3. Grow over night in a warm room on a shaker table.
4. Store any remaining LB in the refrigerator.

Step 3: Mini-Prep of Samples

1. Spin down 1.5 mL of the culture for 30 seconds at approx. 32,000 rpm. Remove and discard the liquid.
2. Suspend the bacterial pellet in 150 microliters of STET(2x) and 150 microliters of sterile water by drawing the liquid up and down the pipette. The Triton is a detergent that breaks down the lipid layer comprising the outer membrane. By placing the cells in a high sucrose environment the fluid flow of the cell goes from internal to external creating mechanical stress. Eventually, due to increasing stress, the inner cell wall ruptures.
3. Add ten microliters of lysozyme (20mg/mL) and boil for 45 seconds. The lysozyme digests the carbohydrate membrane (peptidoglycan).
4. Centrifuge five minutes (32,000 rpm) at room temperature. DNA and RNA will form the pellet.
5. Label a new set of tubes and pipette 300 microliters of isopropanol into each test tube.
6. Remove the liquid from '4' and pipette into the tubes containing the isopropanol. Invert the tube to mix. The isopropanol causes the DNA and RNA to precipitate down from solution.
7. Centrifuge ten minutes (room temperature is adequate, but colder spins tend to yield better results) at 32,000 rpm. Pour off the liquid.
8. Add 750 microliters of cold 70% ethanol. Invert to rinse the pellet. Centrifuge for five minutes (32,000 rpm).
9. Dry the pellet in a speed vac for about 10-15 minutes. If not available leave sample in a hood overnight.
10. Suspend the pellet in 50 microliters of sterile water. Mix by drawing the solution into the pipette and dispensing. (Too much mixing with the pipette will shear the DNA) This solution represents the undigested DNA.

Step 4: Digesting DNA Using a Restriction Enzyme

1. Obtain a microtube and dispense ten microliters of undigested DNA. Add two microliters of Universal Buffer, two microliters of HindIII, EcoRI, or another restriction enzyme to a microtube. Centrifuge briefly to deposit and mix contents. This solution represents the digested DNA. (Refer to "Suggested Table for Dispensing Samples" as a guide)

Step 5: Preparing the Agarose Gel

1. Prepare a 1% agarose solution. For a small gel mix .5g agarose in 49.5 mL of TAE buffer. For a larger gel double the quantities.
2. Microwave the agarose/TAE buffer solution in a flask covered with saran wrap until dissolved (start with 1.5 minutes on high).
3. Pour the solution into a gel tray. (You can tape the edges of the gel tray to get a thicker gel) Once poured insert the comb.
4. Let solidify (approx. 30 minutes) then remove the comb.
5. Place in the buffer box. Make sure the gel is slightly covered with buffer.
6. Extract ten microliters of solution containing DNA, water and Gel Sample Buffer from sample (See reference chart for data). Load the samples into the wells.
7. Attach the cathode (neg. electrode) on the side of the box that is closest to the wells.
8. Run the gel at 100 volts for one hour or until the dye is about 2/3 down the gel.

Suggested Table for Dispensing Samples

Step 6: Staining/Destaining Protocol

1. Stain the gel in ethidium bromide for approximately five minutes.
2. Destain in water (or TAE if gel is to be reused).
3. Photograph gel on UV light next to ruler (5.6, Ğ second).

Solution Concentrations:

TBE (10x) Buffer 1L
108g Tris
55g Boric Acid
50 mL 0.5M EDTA
Mix all in an Erlenmeyer flask with a stir bar. Autoclave.

DEPC H2O 2L
2L nanopure water
100 uL DEPC
Mix and autoclave.

STET (2x) 100 mL
16% glucose or sucrose (32 mL 50%)
100 mM Tris-Cl pH8 (10 mL 1M)
100mM EDTA pH8 (20mL 0.5M)
1% Triton x-100 (1 mL 100%)
nanopure water (33 mL)
Mix and sterilize then filter.

TAE (50x) Buffer 1L
242g Tris
57.1mL Acetic acid
100 mL 0.5M EDTA
Mix all in an Erlenmeyer flask with a stir bar. Autoclave.

EDTA (0.5M) 1L
186.1g NaEDTA.2H20
approx. 20g NaOH
Dissolve NaOH in approx. 800 mL water and pH to 8. Slowly add EDTA while stirring with a stir bar at low heat. Adjust the pH to 8 and autoclave.

1% Agarose Gel
25 mL 1x TAE
2.5g Agarose
dissolve and degas the solution by placing the mixture in a microwave oven for short bursts of approx. 20s

Ethidium Bromide soln. 200 mL
(Considered a mutagen)
Dilute the solution until a light orange tinge develops.

Students are to assign sizes to the DNA bands that they see, using the restriction map of phage lambda DNA as a guide. Students are to record their results in their journals by graphing the samples based on the log of the marker size vs. distance traveled (cm). From this data, students are to determine marker sizes for the bands produced by BamH1.

Questions

1. What is the effect on your gel pattern of using two restriction enzymes.
2. Some bands were more intense (clarity) than others. What do you think the significance of this is?
3. Restriction enzymes are produced by various kinds of bacteria. How do bacteria use the enzymes?

Conclusions

• Summary of Lab Experience: highlight electrophoretic technique utilized
• Explanation of Observations: Based upon your research and general scientific knowledge explain your qualitative data.
• Sources of Error: List three possible sources of error in your lab and how these would effect the outcome.

Data Analysis and Extension:

Each group of four students will be subdivided into two lab stations(two groups of two). The group will be given a sample of DNA that has been digested using HindIII, EcoRI, and BamHI(although others can be used). The small groups of two will run the gel using the same set of conditions in order to compare their results when completed. Each large group of four will be given a sample that contains one similar (EcoR1) or (HindIII) and one dissimilar restriction enzyme. Groups will post their results during a group presentation explaining similarities and differences in the data generated using the poster technique. Each member of the group will be assigned a particular section of the presentation: (1) Summary of lab experience, (2) Presentation of data, (3) Comparison of data, and (4) Sources of error with practical application.

Teacher Guide
for
Electrophoresis of DNA
Using Agarose Gel
(Understanding the Role of Restriction Enzymes)

Answers to Text Questions:

1. Students should see more bands associated with the mixing of the two restriction enzymes.
2. Brian check your pictures
3. Bacteria use restriction enzymes as a means of defense against viruses!

*Note: To make this an easier preparation, companies sell pre-cut DNA with a variety of restriction enzymes. It is a little more costly but can save time!

Marker Sizes
EcoRI---

1. 21266 *(faint bands when heated)
2. 7421
3. 5804
4. 5643
5. 4878
6. 3540 *

HindIII---

1. 27,500 present when unheated
2. 23,130 left arm
3. 9416
4. 6682
5. 4361 right arm
6. 2322
7. 2027
8. 564 faint
9. 125 "
   

Appropriate Agarose Concentrations for Separating DNA Fragments of Various Sizes

Agarose %----Effective Range of Resolution of Linear DNA Fragments (kb)
0.5%____________________________________30 to 1
0.7%____________________________________12 to 0.8
1.0%____________________________________10 to 0.5
1.2%_____________________________________7 to 0.4
1.5%_____________________________________3 to 0.2

* 1 Kb DNA ladder is suitable for sizing linear double-stranded DNA fragments from 500 bp to 12 kb. The bands of the ladder each contain from 1 to 12 repeats of a 1,018-bp DNA fragment. In addition to these 12 bands, the ladder contains vector DNA fragments that range from 75 to 1636 bp.

** There is also a product called the DNA Mass Ladder(patent pending) is suitable for estimating the mass of unknown DNA samples by ethidium bromide staining. The ladder consists of an equimolar mixture of six blunt fragments from 100 to 2000 bp. Electrophoresis of 4 uL of DNA Mass Ladder results in bands containing 200, 120, 80, 40, 20, and 10 ng (470 ng total) of DNA.

Appendix A
Making Gel Boxes Inexpensively

Materials

For each gel box set-up you should have:

two small containers for buffers (plastic is preferred, beakers will suffice)
5" x 5" glass plate (plate glass is preferable)
plastic square to elevate glass gel plate to height of buffer containers
two electrodes (soft graphite pencils available at an art store, pieces of stainless steel or platinum)
soft white paper towels or chromatography paper to act as a salt bridge between wells and gel
Plexiglas cover to prevent drying of gel and keep student fingers out of the way

All gel boxes consist of two wells for buffer solution, an elevated area to place gel plates and two electrodes. Some gel boxes have the gel plate or tray immersed in the buffer solution, but we did not find it satisfactory. Absorbent white paper strips can be placed with one end in the buffer well and the other draped over the edge of the gel plate so that a complete connection is made.

The author found that a small 1-1/2 qt Rubbermaid container was satisfactory. Two small plastic containers (225 mL each) are placed on opposite ends to serve as buffer wells. Any small plastic pieces can be placed between these two wells as a support for the glass plate with the gel. The author used an overturned plastic basket. It is not necessary to place all these smaller components in a larger container, but it leads to stability and safety.

This author has tried graphite pencils as electrodes and found them satisfactory for a short term. If you want to repeat this experiment for many years, obtain stainless steel strips or rods from a hardware store to use for the cathode. Platinum wire is the best choice for anode, but is not needed for the dye separation If you use platinum wire, 0.25mm diameter will suffice and costs about $25.00 for 25 cm. The electrodes chosen can be taped to opposite sides of the larger container. If you have platinum electrodes from a Hoffman apparatus, they should also suffice. Any method to make electrodes secure should be chosen.

Finally, you need absorbent paper to function as a salt bridge between the buffer trays and the gel. Double the paper towels for greater ion flow. Drape the towel over opposite ends of the gel plate. Fill the buffer wells with enough solution to minimize the distance between buffer solution and gel plate. This prevents the towel drying out. The author found inexpensive white paper towels quite satisfactory and much less expensive than chromatography paper. Blotter paper purchased in office supply stores seems to be too resistant to current flow and would not be advisable.

To help students load wells in gel plate, narrow tipped Eppendorf tips are very convenient but you can use microtip plastic Beral pipets or Pasteur pipets with the glass tips pulled out to a thinner diameter.

APPENDIX B
Teacher Suggestions for Electrolysis

Classroom Environment:

Students should work in pairs.

Materials

For each pair of students you need:

one 9 V battery
one battery clip (available inexpensively from Radio Shack in packs of 6)
one pair alligator clips to connect from battery clip to pencils
one acetate sheet
two pencils, each sharpened on both ends to serve as electrodes
approximately 5 mL of 0.2M CuCl₂
paper towel to clean electrodes

The battery set-up is more sturdy if you can cut the alligator clip connectors available from Radio Shack in half and solder the cut ends to the battery clip as shown in the lab diagram. Once prepared, class sets last for years. (The author has used the same set for 8 years with no problems. The batteries last 5-6 years.)

Answers to Analysis Questions:

1. Copper is the solid deposited.
2. Copper ions are positively charged and are attracted to the negative electrode because opposite charges attract.
3. The substance released at the anode is chlorine gas. The chloride ion traveled to the electrode because it had an opposite charge.

Answers to Extensions

1. Positively charged proteins would move toward the cathode. Negatively charged proteins would move toward the anode.
2. Ions could be slowed by using a substance that traps them such as a gel or tangled polymers. Proteins with greater molecular mass would move more slowly. (Use the analogy of trying to retrieve a pebble from a bowl of clear soup or from the bottom of a plate of cooked spaghetti.)
3. DNA is also separated using gel electrophoresis. The DNA chain is negatively charged because of the phosphate groups and travels toward the anode.

Appendix C
Teacher Instructions for Micropipet Practice Activity

Abstract

Students develop technique in micropipets use while preparing four colors from four basic food dyes.

Instructional Strategy

Most separations of proteins are on a very small scale and use micro amounts of liquids. This requires ability to manipulate hand-held instruments called micropipets. This activity will familiarize students with their use and offers a quick method of determining if theyıre using them properly.

Materials:

micropipets, plastic microfuge or vortex mixer, (Eppendorf) microtubes, pipet tips, glycerin, distilled or de-ionized water, filter paper, food coloring dyes (red, green, blue & yellow), 6' x 6" Styrofoam sheets to act as microfuge holders, vortex mixer or plastic toothpicks for mixing

Teacher Preparation

1. Stock dye solution preparation for 8 class sets
   1. Mix 4.0 mL desired food coloring and 4.0 mL of distilled water
   2. Add 16 uL of glycerin to mixture and mix thoroughly. (A vortex mixer is preferred)
   3. Divide each colored solution into 8 labeled microtubes.
2. For each group of students prepare a kit consisting of:
   1. A Styrofoam sheet or piece of foam to act as a micro test tube holder containing:
      • 4 empty microfuge tubes;
      • 4 labeled microfuge tubes each with one of the four basic colors
   2. 1 micropipet
   3. filter paper

Special Notes:

1. Micropipet tips can be rinsed and reused.
2. Teal, rose orange and chartreuse colors will be prepared.
3. Comparison of the spots on the filter paper indicates the student's abilities to pipet 3 uL correctly.
4. Prepare a standard so you can compare their teal, rose, orange and chartreuse colors. This also indicates their ability to use the micropipets.

Appendix D
Teacher Suggestions for Gel Preparation

Short cuts:

1. Prepare agarose solution earlier and keep warm (70^oC for agarose, 60^oC for Knox gelatin) in a water bath on a hot plate. If gel cools too much it will solidify. If it is too hot, the gel takes too long to cool and harden.
2. Knox gelatin can be substituted for agarose using dyes only. If you are substituting, a 3% - 5% is needed for gel formation (one package per 1 cup of water is a 2.9% solution. This gel will revert to the sol (liquid) state more quickly if too much voltage is used during electrophoresis unless you have a method of cooling the gel. If you use this gel DO NOT EXCEED 100 V FOR ANY REASON. If the gel melts, an electrical short can occur in the system. (This author used 3% solutions, but 5% has a lower chance of melting.)
3. Combs can be made from Plexiglas. Each tooth is 6.25mm wide. The thickness of the Plexiglas on commercial combs is 0.75mm. A comb can be cut from high density polyethylene gallon milk bottles if no Plexiglas is available. These do make very narrow slits, but will suffice. Remember that the teeth should not go completely through the gel. Diagram follows.

Appendix E
Teacher Dye Lab Instructions

Instructional Strategy

Classroom environment: High school chemistry

This type of activity embodies discovery learning, so resist the urge to say too much at the beginning. Be sure students understand technique. Students may prepare a mixture of dyes for another group to identify.

Teacher advance preparation for gel electrophoresis with dyes

• Equipment needed for class of 32 (8 groups of 4)

  DC Electrophoresis power supplies (100 V or greater) Some can be shared by 2 or more groups
  balances
  hot plate or microwave to heat gel
  8 glass plates about 5" x 5" 8 acetate sheets and markers or 8 sheets of blotting paper
  8 heat resistant gloves (optional)
  8 electrophoresis gel boxes (commercial or constructed as in Appendix A)
  assortment of dyes (Appendix F)

Teacher Prep time - about 2-3 hours
Prepare dyes as listed in Appendix D or purchase from commercial sources listed in Appendix E

Running buffer, 1X TAE (0.4M Tris-acetate; with 0.001M EDTA)
This is more conveniently prepared by using a 50X TAE solution and diluting 40mL of the 50X stock with water to a final volume of 2 liters. Many gel boxes use about 125 -200 mL of 1X buffer. The buffer may be reused several times if mixed completely between runs.

Recipe for 50X TAE concentrate

242 g Tris base available from Sigma Chemical Co., Flinn or Frey
57.1 mL glacial acetic acid
100 mL 0.5M EDTA, pH 8

(This stock is made from disodium EDTA and pH balanced using a pH meter or pH paper and HCl.) Make volume 1 liter with deionized or distilled water. This reagent solution may also be purchased.

You may wish to prepare some practice gels ahead of time and use one class period for students to practice pipetting and loading wells in these gels.

If students are using micropipets and tips they need prior instruction and practice as indicated in the strategy. If using pulled out Pasteur pipets or micro tip Beral pipets, take time to instruct them in their use to obtain very small sample size.

Answers to Analysis Questions

1. Answers will vary. Dye directions indicated in Appendix F.
2. Answers will vary. Dye directions indicated in Appendix F.
3. More samples travel to the + electrode. They are often anions of salts.
4. Red dyes are supposed to be different so the distance traveled should vary.
5. Students may be surprised that yellow dye is yellow & red and that blue is blue with a little red. Green is yellow and blue.
6. If the electrophoresis runs long enough they can distinguish mixtures. Otherwise not.
7. The properties of charge and molecular mass are used to separate them.
8. Gels with smaller holes do better separations. More concentrated gels have smaller holes. Running electrophoresis for a longer time helps separation is gel plate is long enough to use. (Agarose gels will give better results than the Knox gelatin.)

Appendix F Dyes for Dye Electrophoresis

The following dyes are suggested. Some are commonly used as acid-base indicators and others are biological stains. Try to use some that are cationic and some that are anionic.

Other suggestions: mercurochrome, food colors, inks, Easter egg dyes, tie dyes, flower extracts, cabbage juice, beet juice, berry juice, iodine. This author was unsuccessful with a commercial dye, Rit dye. Canned beet juice worked very well and students should see two colored bands, bright pink and pale orange. To use any of the former, try to prepare a relatively dark extract by pulverizing plant material in mortar & pestle or in a blender with minimum amount of water. Strain before use.

Student groups can make a simple mix of dyes but mixtures of + & - dyes forms precipitate readily. Mixes should all be negatively or all positively charged.

Prepare dyes in a 0.25% solution (25 mg/ 10 mL). Add 1 mL glycerin to make mixture more dense to help loading the gel wells. Preparation of dye mixtures requires care to avoid a dye mess and avoid inhaling powders. Use an apron and gloves and work in a well ventilated area. Dye solutions can be purchased from commercial sources listed in Appendix H.

Appendix G
Teacher Strategy
Determination of Molecular Weight of Unknown Protein

Instructional Strategy

This problem solving activity provides classes of different abilities to determine the molecular weight. For students with few prerequisite skills, provide the activity as given on page 19 which gives the distance data Advanced classes should be provided only with the diagram below which simulates actual data obtained on gel electrophoresis of those proteins. Students must measure the distances themselves just as a research chemist would do. They will obtain a greater margin of error. The light lines are added to aid measurement, but would not be on any gel. The myoglogin band is curved in a "smile" to simulate what sometimes occurs on the edges of the gel. Also a second light band is given for hemoglobin which might get fragmented into halves.

Appendix H
Sources of Materials

Bio-Rad
Source of proteins, micropipet & tips
Pipet tips w/ fine tip for injecting in wells, Cat. # 223-9915\

Kaleidoscope prestained standards, Cat # 161-0324; $90 for 500 m L

Flinn Scientific
P. O. Box 219
Batavia, IL 60510-9958
Phone 800-451-1261

Source of kits

• Molecular weight of proteins with 3 proteins, SDS & agarose, Cat # FB0308; $96.25
• Agarose gel kit with dyes; Cat. # FB 0300; $56

Source of dyes: purchased separately

• orange G, Bromocresol green, bromocresol purple, phenol red, methol red, methylene blue, crystal violet

Source of 50X TAE electrophoresis buffer, concentrated

• agarose solution, melt & pour
• electrophoresis stains

Frey Scientific, 905 Hickory Lane, P. O. Box 8101, Mansfield, OH 44901-8101,

• PH 800-225-FREY
• Electrophoretic Properties of Native Proteins Kit, #F18888, $85
• Introductory Electrophoresis Kit, #F18879, $50 (Includes 6 dyes, gel, agarose & buffer)
• Molecular Weight Determination of Proteins Kit, # F18887, $85

Source of Tris-glycine, SDS buffer, Polyacrylamide, gels reagent, coomassie stain for proteins, protein grade agarose,

Video on electrophoresis

Sigma Chemical
P. O. Box 14508
St. Louis, MO 63178-9916

Source of reagent grade agarose, proteins & dyes, platinum wire