7 Electrophoresis. µ proportional to Q

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1 7 Electrophoresis Objectives: A) To perform agarose gel electrophoresis of the proteins isolated in last week's experiment and B) to interpret the banding patterns produced by these proteins. Introduction: Electrophoresis is the migration of charged molecules in an electric field. It is an important analytical technique for the separation of biomolecules (proteins, amino acids, nucleic acids, lipids). The electrophoretic mobility of a charged particle is the extent of movement of that particle in an electric field. Electrophoretic mobility of small molecules is greater than that of large molecules with the same charge density. In a medium of constant viscosity and voltage, the mobility of a particle is directly proportional to its charge to size ratio: µ proportional to Q/r where- µ = electrophoretic mobility Q = net charge of the molecule r = radius (size) If the molecular size of the molecules in a sample is approximately the same, the electrophoretic mobility is directly proportional to the net charge: µ proportional to Q Usually, the ph of the gel is high enough (often above ph 8) so that nearly all proteins have net negative charges and move toward the anode when the current is switched on. Molecules of similar size and charge will migrate through the gel as a single band. In gel electrophoresis, a semi-solid gel (matrix) is used as the supporting medium. The two gels of choice for separating proteins or nucleic acids, namely agarose and polyacrylamide, are both cross-linked polymers. Thus, both gels are porous in composition. The advantage gained with this porosity is that pore size can be controlled to yield gel matrices with a wide variety of uniform pore sizes. An agarose gel is a semi-solid medium that represents a polysaccharide obtained from agar. It is composed of a linear polymer of D-galactose that

2 is cross-linked to galactose 3.6 anhydro L-galactose. Agarose gels can provide very large pore sizes. Because of the large pore sizes, agarose is the medium of choice when analyzing nucleic acids, the largest biomacromolecules in size. Additionally, it is the preferred electrophoretic medium when one analyzes the structure of proteins or protein complexes in their native conformation. Agarose gel electrophoresis of a protein is commonly used to determine a) its isoelectric point or b) whether it has quaternary structure. Polyacrylamide gel electrophoresis (PAGE) is the method of choice when one examines denatured proteins and/or nucleic acids, or relatively small biomacromolecules such as peptides and DNA fragments. A polyacrylamide gel consists of a polymer of acrylamide [CH 2 =CH CONH 2 ] that is occasionally cross-linked with bis-acrylamide (N,N'-methylene bis-acrylamide) [CH 2 =CH CONH CH 2 NHCO CH=CH 2 ]. TEMED (N, N, N', N'- tetramethylene diamine) and ammonium persulfate are needed to initiate the polymerization of acrylamide and bis-acrylamide. The pore size is determined by the total acrylamide concentration. Very small pore sizes can be obtained with polyacrylamide gels by varying the concentration of acrylamide (the bis-acrylamide concentration usually remains constant). The degree of resolution of a mixture of molecules will depend on, to a large extent, the concentration of the matrix. Consequently, increasing the percent (w:v or v:v) of the matrix, either acrylamide or agarose, enhances the separation or resolution of small molecules. On the other hand, low percent matrix gels increases the resolution of larger molecules. Common Electrophoresis Patterns * Direction of migration Lane 1 is the pattern indicative of a well-resolved mixture of proteins except for (*). The band indicated by the (*) actually represents several unresolved proteins. The smile in Lane 2 is indicative of a sample that was electrophoresed too quickly. The frown indicated +

3 in Lane 3 indicates uneven heating of the gel during electrophoresis. Lane 4 represents the pattern observed when the protein sample is overloaded (i.e., too much protein was loaded). Lane 5 indicates a degraded protein sample. Lane 6 represents a pure protein sample. Note: The banding pattern exemplified in the above figure will be observed regardless of the electrophoretic medium. Denaturing PAGE is generally performed if, for example, one wants to ascertain the relative molecular mass of a protein or its quaternary structure. In denaturing PAGE, the detergent sodium dodecyl sulfate (SDS) is used as the denaturant. This type of electrophoresis is generally referred to as SDS-PAGE. When SDS-PAGE analysis is used to determine the relative molecular mass of a protein, one must compare the migration of the protein under analysis to that of protein standards. The relationship between migration distance and molecular mass is linear when analyzed via a semi-log plot: Logarithmic Relationship between the Molecular Mass of a Protein and Its SDS-PAGE Electrophoretic Mobility Molecular mass (kd) Relative mobility The location of a protein that is electrophoresed through a gel can be ascertained with the aid of a dye. The most common method of visualization is by staining with a solution containing Coomasie Brilliant Blue, the same dye used in the Bradford reagent. After excess dye is removed by washing the gel in a destain solution, the proteins appear as bands in the gel matrix. Coomasie staining very easily detects microgram quantities of protein loaded into a gel. A much more sensitive form of protein detection relies on silver. Silver staining, although much more expensive than Coomasie staining, is a necessity when the protein of interest is present at sub-microgram quantities. Coomasie staining is done with both agarose and polyacrylamide gels, whereas as silvers staining is reserved for the latter.

4 Capillary electrophoresis is an analytical electrophoretic technique used when a very small quantity of sample is available. In this method, electrophoresis is carried out in very thin capillary tubes. The use of these thin tubes facilitates rapid heat dissipation. Consequently, very high electric fields are used resulting in both high resolution and separation times of minutes rather than hours. Paper electrophoresis is the most popular procedure used to fractionate amino acids and peptides. Low molecular weight proteins are not separated by paper electrophoresis because the cellulose matrix limits their resolution. Cellulose acetate electrophoresis of proteins is the method of choice in clinical laboratories that test for sickle cell anemia. In today s experiment, we are going to electrophorese casein and BSA, the proteins upon which you conducted ammonium sulfate fractionation, using polyacrylamide as the gel matrix. The gel will be stained, and then destained. The migration of these proteins will be compared to a mixture of standards (indicated in the following table) and their respective molecular weights estimated. Bio-Rad Kaleidoscope prestained Standards Protein Color* Size Myosin Blue 216,000 β Galactosidase Magenta 132,00 BSA Green 78,000 Carbonic Anhydrase Violet 45,700 Soybean trypsin inhibitor Orange 32,500 Lysozyme Red 18,400 Aprotinin Blue 7,600 Procedures: A) SDS PAGE 1. An 8% SDS PAGE gel has already been prepared. For reference purposes the recipe is listed below: Solution components Resolving (or separating) gel Stacking gel H 2 O 2.3 ml 1.4 ml

5 30% Acrylamide 1.3 ml 0.33 ml 1.5 M Tris ph ml M Tris ph ml 10% SDS 0.05 ml 0.02 ml 10% ammonium persulfate 0.05 ml 0.02 ml TEMED ml ml 2. SDS PAGE is a denaturing gel. The proteins must be boiled in the presence of SDS to denature them. Thus we need to add 5 µl of Laemmli loading dye solution. This contains a dye, used for determining how long to let a gel run. The dye is smaller than the proteins, thus runs faster. As long as the dye has not run off of the gel we can be sure we have not lost the protein. The Laemmli also contains a reducing agent to reduce the disulfide bonds. 3. Boil the samples, molecular weight marker as well, for 4 minutes. 4. Remove from the boiling water and place on ice. 5. Load the gels into the apparatus. The short plates need to be on the inside. 6. Fill the apparatus with 1 X SDS running buffer; fill up to the top of the tall glass plate, inside and outside. 7. Load the samples, any order is fine AS LONG AS YOU RECORD THE ORDER IN YOUR NOTEBOOK. People usually load the markers on the left. Loading the sample can be difficult. Place the pipette tip into the buffer and touch the back plate. Slowly raise the tip until you feel the tip move forward to the tall plate. Place the tip over the well and SLOWLY pipette out the sample. The sample should sink to the bottom of the well. 8. Once all the samples, and markers are loaded start the gel at 100 volts. After 20 minutes increase the voltage to 150 volts. 9. After the gel has run for about 1 hour turn off the power supply and remove the gel. Carefully, using a spatula or razor blade pry the glass plates apart. This is done by inserting the tip of the blade between the plates and using a GENTLE twisting action. 10. The gel will stick to one plate, immerse the plate and the gel in the running buffer and carefully remove the gel from the glass. The gel has a tendency to stick to the plate and rip. T 11. Cut one corner of the gel so you know the orientation, I usually cut the lower left hand corner, the side with markers, which I usually run on the left. There is nothing magical about this orientation. What is vitally important is that YOU know what sample was loaded where.

6 12. Place the gel in staining solution and gently shake. Staining should take about 15 minutes. 13. After 15 minutes, pour the stain in the bottle labeled Used Stain. 14. Rinse the gel with deionized water to remove the staining solution. 15. Place gel in de-stain solution and shake. 16. Once the de-stain solution gets too blue remove the blue de-stain and replace it with fresh de-stain. 17. a. Determine the distance each protein band in the standard lane migrated from the well by using a ruler to measure from the bottom of the well to the center of the band. Record the distance in cm. b. Use Microsoft Excel to plot log MW of the standards versus the distance migrated. c. Using linear regression analysis, determine the molecular weight of both casein and globulin by plugging their migration distances into the generated equation. A plot of pi vs. distance yields a line with a negative slope. Why? On your image, you must identify of the sample loaded into the well. Your report must also include a discussion of the sample s integrity based on the shape of the band (i.e., narrow, broad, smile, frown, smear, single band, multiple bands, etc.), and the sample s estimated molecular weight. Does this experimentally determined molecular weight match the estimate given on page 42? Make a photocopy of the gel or sketch it in your notebook: One partner needs to turn in the actual gel with your lab report, the other partner needs to turn in a photo copy of the gel.

7 Electrophoresis STUDY GUIDE 1. What is electrophoresis? 2. In a medium of constant viscosity and voltage, what determines the electrophoretic mobility of a substance? 3. When is the electrophoretic mobility of molecules in a sample directly proportional to their net charges? 4. What determines pore size of a polyacrylamide gel? 5. Explain, in detail, how you would prepare 40 ml of 1% (w:v) agarose gel for an electrophoresis experiment. 6. A sample composed of a mixture of proteins myoglobin, β-lactoglobulin B, cytochrome c, and bovine carbonic anhydrase yielded one of the two patterns diagrammed below after agarose gel electrophoresis at ph 8.3 and stained with Coomasie. The pi of each protein is indicated in the following table Protein pi Carbonic anhydrase (bovine) 6.5 Cytochrome c 9.6 β-lactoglobulin 5.10 Myoglobin 7.00 a. Identify the correct electrophoretic pattern (A or B). b. Identify each protein. Hint: To solve this problem, use the equation Q = pi - ph. A. B. 2 proteins with same net charge - + The well - + The well

8 7. Why do most proteins migrate towards the positive electrode? 8. Suppose you have 10ng of your favorite protein. What type of electrophoretic analysis should you run if you need to determine the relative M r of your protein sample? Why? 9. Why is the detergent SDS a denaturant and not a degradative agent? 10. You have loaded 2ng of your protein into an agarose gel to determine its pi. Upon completion of electrophoresis followed by staining with Coomasie for an hour and overnight destaining with constant agitation, only the standards were visible. Why?

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