INTRODUCTION
    Gel electrophoresis is a separation technique which is often used to separate large molecules such as proteins or nucleic acids (RNA or DNA) which may have molecular masses in the range from thousands to millions of daltons.  The separation in electrophoresis is based on two characteristics of the molecules being separated -- electrical charge and size.  When a charged molecule is placed in an electric field, it will move toward the electrode with the opposite charge, in much the same way that opposite poles of a magnet are attracted.  If the molecules were just allowed to move freely through solution, it would be difficult to separate them on this basis.  So we force the molecules to move through a polymeric gel, which is made up of a tangled microscopic meshwork of fibers.  Thus, the electric field causes the charged molecule to move, but the molecule must pass through the gel meshwork, which tends to slow its movement.  Thus, if two molecules have a constant ratio of size to charge, and one is larger than the other, the smaller one will move through the gel faster, and if we stop the electric field and look at how far the two molecules have moved, the smallest one will have moved farther than the larger one.  It turns out that gels can in this way separate a fairly large range of sizes of molecules, so that they will be arranged in order of their size, with the smallest molecules moving the greatest distance, and the largest the smallest distance.

    The essential requirement for such a separation on the basis of size is that the ratio of charge to mass must be constant. This is not normally true for proteins, because each type of protein is made up of different amino acid subunits, and the charge of the whole protein molecule depends on which particular amino acids make up that protein, and also on the pH of the solution containing the protein (because the charge on an amino acid depends on the pH, and different amino acids respond to pH in different ways.)

    Another problem with proteins is that they are normally folded up into a specific three-dimensional shape, which will affect how they move through the gel meshwork.  So both the natural charge of the protein and the natural shape of the protein will affect how the protein moves through the gel meshwork.  Sometimes a scientist may want to use exactly those characteristics as a basis for separating some proteins from others.  However, for our purposes, what we would like to do is to eliminate both the natural shape and natural charge as factors in the separation, so that we can separate the protein molecules just on the basis of their molecular weights.  In order to do this, we can add a strong detergent which surrounds all parts of the protein chain and causes the protein to unfold into a long string, which will be completely surrounded by the detergent molecules.  Because the detergent has a strong negative charge which completely overpowers the natural charge of the protein, the protein will now have a constant ratio of charge to molecular weight, which is completely independent of which amino acids make up the protein.  The detergent which is usually used to do this is called sodium dodecyl sulfate, abbreviated as SDS.  (Dodeca is Latin for "twelve", so the "dodecyl" part of the name is just a way of saying that the carbon chain of the detergent is 12 carbons long.)

    The kind of gel which we will be using is called polyacrylamide, because it is a polymer of acrylamide. The microscopic strands of acrylamide in the gel are connected by acrosslinking molecule, called N, N'-methylene-bis-acrylamide, or bis for short. The structure of acrylamide and bis monomers are shown in Figure 1, and a portion of the polymerized network is shown in Figure 2. Both acrylamide and bis are very toxic to nerve tissue, and they are skin irritants as well. However, once they have been polymerized, they lose their toxic properties.

    N,N'-methylene-bis-acrylamide (bis)
Figure 1.  Structures of
acrylamide and bis monomers
 

    We want to separate the protein bands as far apart as possible, but we don't want them to start running off the end of the gel.  Unfortunately, most proteins are not colored, so we can't see how far they have moved through the gel.  This problem can be solved by just adding to our sample mixture a colored dye that moves faster than any protein in our sample.  Such a dye is called a tracking dye, because it allows us to track the progress of our separation, and bromphenol blue is the dye most commonly used for this purpose.

    A second problem which arises from proteins not being colored is that we can't see the position of all those neat concentrated bands of protein after our separation is finished. This problem can be solved by staining the separated proteins with a dye which sticks tightly to proteins, but binds much more loosely to the polyacrylamide gel material.  To make sure our separated proteins don't move around in the gel after the electrophoresis is finished, we will also "fix" them to the gel by treating them with methanol and acetic acid.  Then we will stain them with a blue dye called Coomassie Blue.  Both the gel and the proteins will be stained at first, but then when we remove some of the stain by a process known as destaining, the dye will be almost completely removed from the polyacrylamide gel material, but will remain stuck to the protein bands.

    The result will be a pattern of protein bands, which will result in something which resembles a supermarket barcode.  Each band in the pattern will represent a protein molecule of a particular molecular weight, and to some degree this pattern of proteins will be characteristic of the source from which the proteins came.

    We are going to use this to distinguish genuine crab meat from imitation crabmeat.  As you know, crabs are somewhat difficult to obtain, and hence expensive.  However, many kinds of fish are much less expensive, and it is possible to treat certain types of fish meat so that the final product closely resembles genuine crab, but is much less expensive to produce.  Usually artificial crab meat is pollack that has been shredded, washed of oils and natural flavors (which would give away the real nature of the meat), then mixed with glycogen, glucose, crabmeat extract, artificial flavors and preservatives, and colored and shaped to resemble real crab legs.  However, you might expect that the pattern of bands produced by electrophoresis of an extract of proteins from real crab meat might be different from that resulting from altered pollack meat, despite the tricks that have been used to fool the eye and the palate.  In this lab, we will see whether this is really so.

REFERENCES
    Anthony T. Andrews. 1981. Electrophoresis: Theory, Techniques, and Biochemical and Clinical Applications. Oxford: Clarendon Press.
    Bio-Rod Laboratories. Mini-Protean II Dual Slab Gel Cell Instruction Manual.
    Freifelder, David. 1982. Physical Biochemistry, 2nd edition. San Francisco: W.H. Freeman and Company.
    Gasque, C. Edward. 1989. A Manual of Laboratory Expenences in Cell Biology. Dubuque, Iowa: Wm. C. Brown Publishers.
    FisherBiotech Dual Mini-Vertical Electrophoresis System Instruction Manual, version 1.1 (February, 1988)
    Hoeffer Scientific Instruments Catalog. (Includes "Basic Techniques and Exercises in Electrophoresis ", a mini-lab manual on electrophoresis. )
    Hames, B. D. and Rickwood, D., eds. 1981. Gel Electrophoresis of Proteins: a Practical Approach. London: IRL Press Limited.
    Laemmli, U. K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-85. (This is the original reference for the Laemmli discontinuous SDS-PAGE electrophoresis technique).

    The proteins are put into SDS which is a powerfully negative detergent which binds to the proteins causing them to unfold, and the individual proteins are released from their associations with other proteins or lipids (especially transmembrane proteins). The SDS also coats the surface of the protein causing it to have a negative charge which is  repulsed from the negative electrode of the gel and attracted to the positive electrode.

    2-Mercaptoethanol, which is in the treatment buffer, is added to break any s-s linkages and allows multisubunit molecules to be analyzed separately.
 

    One problem with this one dimensional gel is that many of the bands are closely spaced or may even overlap and thus can resolve only relatively small numbers of proteins.  A 2- Dimensional SDS-PAGE gel can be used to resolve more than 1000 different proteins. This technique combines separation using electrical current and a pH gradient.  The proteins will reach their isoelectric point in the pH gradient.
 

Figure 3.  Separation of molecules by isoelectric focusing

Two Dimensional SDS-PAGE

    You can then isolate the one protein you are interested in and digest the protein into smaller fragments by using enzymes (proteases) or chemicals (cyanogen bromide).  Then use a 2-D SDS-PAGE to separate the fragments. The pattern displayed by this gel is known as a protein's "fingerprint" and is unique to that protein. (This process was developed as a way of comparing normal hemoglobin with the mutant form of the protein found in patients suffering sickle cell anemia, which is the difference of only one amino acid).

    Next is to sequence one of the short amino acid sequences. To do so, the first peptide is exposed to a chemical that forms a covalent bond only with the free amino group at the amino terminus of the peptide. This chemical is then activated by exposure to a weak acid so that it cleaves the peptide bond between the amino acid and the rest of the polypeptide. The amino acid can then be identified by chromatographic methods. This whole procedure is now done by machines called Amino Acid Sequencers.

    Now that you know a short amino acid sequence (only 20 amino acids are required), a DNA probe can be made to find the gene, then sequence the rest of the protein from the DNA sequence.

DATA SHEET 2    Another data sheet to record the sample identities and lanes loaded.

1.  Describe below the purpose for doing this lab.
 
 
 
 

2. Define the following terms:

 a. amino acid
 b. protein
 c. polypeptide
 d.  µL

4. What is the purpose of heating the protein sample?
 
 
 
 

5. What do you think would happen to the pattern of bands on your gel if you did not heat up your protein sample?
 
 
 
 

6. Why is SDS added?
 
 
 
 

7. How does the treatment buffer work on the proteins?
 
 
 

8.  What gets separated from the complex mixtures using the polyacrylamide gel electrophoresis?
 
 
 
 

9.  How does the electrophoresis box move the proteins down the gel?
 
 
 

10.  What does the band at the bottom of the gel indicate after it has been run?
 
 
 
 

11.  What would occur if you increased the voltage in the electrophoresis box while running the gels?
 
 
 

12.  How does one protein differ from another?  Describe at least four ways.

a.__________________________________________________________

b.__________________________________________________________

c.__________________________________________________________

d.__________________________________________________________

14.  How could you get an estimate of the size or weight of a particular polypeptide?  Explain your answer.
 
 
 

15. Of all of the protein samples examined (by the whole class), which two protein samples have the most similar banding pattern?
 
 

16. What might a similar banding pattern suggest?
 
 
 

17.  Which fish protein was closest to sole?  Explain why?
 
 
 

18.  Which fish protein was the most different?
 
 

19.  Do you think this has anything to do with different flavors?
 
 
 

20. Using the size (molecular weight) of the standards below, estimate the size (weight) in daltons for each polypeptide in the protein sample assigned by your teacher.
 

21. EQUIPMENT CHECK
 Demonstrate the proper techniques for the following:

 Eppendorf micropipettor               ____________________
 Centrifuge                                     ____________________
 Loading electrophoresis gel           ____________________

22. Draw out below NEATLY (using a ruler) the results of your 10 Lane Gel.  Draw all ten lanes and label each lane.
 

STEP	PROCEDURE		PURPOSE	"VISUAL"
1				Protein composed of 2 polypeptide chains
2	PROTEIN SAMPLE
	10 µL
3	TREATMENT BUFFER
	10 µL
4	5X LOADING BUFFER			Tracking Dye Added
	10 µL
5	CENTRIFUGE			Mix
	10 µL
6	BOILING WATER BATH
	1 min.
7