BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

LAB 3 PAGE DETERMINATION OF PROTEIN FINGERPRINTS STUDENT GUIDE Information from an NSF sponsored workshop was used in adapting this lab: Genomics: from Mendel to Microchips, taught by the Partnership for Plant Genomics Education at the University of California Davis, July 2005. The lab was originally adapted from Biotechnology Explorer ™ Protein Fingerprinting Instruction Manual Bio-Rad DNA Fingerprinting – Partnership for Research and Education in Plants.

GOAL The goal of this lab is to separate proteins by polyacrylamide gel electrophoresis and to analyze the data using a standard curve graphed using MS Excel. OBJECTIVES After completion, the student should be able to 1. Perform a common type of plant protein extraction 2. Perform the technique of SDS-PAGE. 3. State the purpose for SDS-PAGE in the research laboratory. 4. Explain how proteins will move through SDS polyacrylamide gels when placed in an electric field. 5. Explain the need for denaturation and reduction when separating proteins by size. 6. Estimate the molecular weight of a protein using proteins of known molecular weights as standards. 7. Analyze electrophoresis data using manual and Excel graphing. TIMELINE This lab will take 2 laboratory periods: DAY 1: Prep for the lab, extract plant proteins, run SDS PAGE, stain and destain DAY 2: Graph results using MS Excel and analyze BACKGROUND Proteomics is the study of the complete set of proteins present in an organism. The number of proteins in an organism far exceeds the number of genes, which is due in part to molecular mechanisms of gene control. These processes include shuffling of DNA to form different genes (such as that seen in antibody production), post transcriptional control, and post translational control of protein production. Proteomics is a growing field that employs sophisticated molecular techniques such as two dimensional gel electrophoresis and mass spectrometry. Understanding the basis of protein separation by polyacrylamide gel electrophoresis is the first step in understanding proteomics. Electrophoresis is a technique that separates charged molecules in an electric field. Negatively charged molecules migrate in an electric field toward the anode; positively charged molecules move toward the cathode. In polyacrylamide gel electrophoresis (PAGE), molecules move through a porous gel matrix that separates molecules on the basis of both charge and size. This migration is complicated because both the size (mass) and the net charge of the molecule contribute to the migration. Proteins can have different

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charges because the side chains of many amino acids carry a charge. Proteins move through the gel based on their charge to mass ratio. That is, the higher the negative charge, the faster the protein migrates; conversely, the larger the size, the slower the protein migrates. The separation of proteins in polyacrylamide gels on the basis of their charge to mass ratio is called non-denaturing or native gel electrophoresis. This technique is most commonly used when the native conformation and activity of the protein must be maintained. Many proteins of different sizes have similar charge to mass ratios; these proteins would migrate very similarly in a polyacrylamide gel, and therefore would not be resolved. Another problem with native gel electrophoresis is that many proteins consist of multiple subunits that make the molecule too large to separate easily by polyacrylamide gel electrophoresis. These problems can be overcome by another technique called denaturing gel electrophoresis, or SDS polyacrylamide gel electrophoresis (SDS-PAGE). In this technique, proteins are treated with a strong anionic (negatively charged) detergent called sodium dodecyl sulfate (SDS) that binds to proteins in proportion to the size (mass) of the protein (about one molecule of SDS per amino acid). That is, a protein of 20 kD would bind twice as much SDS as a protein of 10 kD. In addition to the influence of mass on protein movement through polyacrylamide, protein shape can also affect the migration rate. To separate two proteins strictly on the basis of size, they must also have the same shape. When protein samples are denatured by heating and treatment with reducing agents such as -mercaptoethanol (that break disulfide bonds between two cysteine amino acids), the polypeptides that make up the protein separate, unfold, bind SDS and assume a rod-like structure. Since the length of these rod-like molecules is proportional to the size of the polypeptide, denatured SDS polypeptides migrate in polyacrylamide gels primarily on the basis of size. Proteins separated by SDS-PAGE can be compared to denatured polypeptides of known size to determine their mass. Polyacrylamide is a synthetic polymer or chain of acrylamide monomers. These acrylamide chains can be crosslinked to each other by the addition of bisacrylamide during the polymerization reaction. The bisacrylamide crosslinks cause the chains to form a mesh-like structure, in which the holes of the mesh represent the pores that retard protein migration in the gel. At higher acrylamide or bisacrylamide concentrations, the mesh becomes tighter with smaller pores that more strongly retard the migration of proteins. Proteins can be separated on polyacrylamide gels of a consistent concentration determined by the sizes of the proteins to be separated. Large proteins require a lower concentration of acrylamide in the gel. Acrylamide concentrations in the range of 10% to 15% separate proteins of about 12,000 to 70,000 daltons. In many instances, separation of proteins can be improved by the addition of another layer of acrylamide of a different pH atop the separating gel. This top layer, called a stacking gel, has large pores that allow rapid migration of the proteins until they reach the boundary where the separating gel begins. The protein migration abruptly slows, and the proteins stack, so that they enter as a thin zone at the surface of the separating gel. The pH and concentration differences between the two gels result in well defined, narrow protein bands that are better resolved making analysis easier. A gradient gel is used to separate proteins of widely different sizes while also separating those of similar sizes. The highest concentration, which

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retards small proteins, is at the bottom of the gel and the lowest concentration, for larger proteins, is near the top. In addition to the concentration of acrylamide in gels, the buffer system affects the migration of the proteins. A discontinuous buffer system, in which the buffer and the gel have dissimilar ions, helps to stack the proteins at the boundary of the separating gel. After the proteins stack, they migrate through the separating gel with a constant pH and voltage, allowing resolution by the sieving action of the acrylamide pores, thus separating the proteins by size. Separated proteins can be visualized by staining with Coomassie stain, a blue dye that binds strongly to proteins. Before the proteins can be stained, they must be bound or fixed to the gel matrix with acetic acid. The coating of SDS must also be removed from the proteins so they are accessible to the Coomassie blue dye. SDS is removed from the gel and the proteins with methanol or isopropanol. The Coomassie stain contains methanol, acetic acid and Coomassie blue. After the proteins are stained, the gel must be "destained" so the protein bands become visible. The destain solution contains lower percentages of methanol and acetic acid. Coomassie blue can detect as little as 0.1 g of protein per band. In this laboratory SDS-PAGE is used to analyze plant protein samples. The proteins will be resolved on a precast gel (check the manufacturer for the concentration) using a discontinuous buffer system. The molecular weights of the major plant proteins isolated by the class will be calculated based on their migration relative to the protein standards of known sizes. The proteins of the molecular weight standards are prestained with conjugated dye. Protein samples are mixed with a loading buffer/dye solution that contains SDS and -mercaptoethanol to denature the proteins. Sucrose or glycerol is included in the loading buffer to increase the density of the sample so it can be loaded on the gel, and bromophenol blue is included to help visualize the progress of migration during electrophoresis. Most vertical gel chambers accommodate two gels at the same time. In this case, 2 teams will work together to prepare samples and run two gels simultaneously on one gel chamber. Three unknown samples will be run with molecular weight protein markers. LABORATORY OVERVIEW In this lab, young plants of various types will be used to extract proteins from different developmental regions, i.e., leaves, stem, flower, sepals and roots. The crude protein extracts will be run on an SDS polyacrylamide gel, which will be stained, destained and analyzed for different patterns. Prominent protein bands will be sized using a standard curve, and conclusions will be drawn regarding the possible identification of these proteins.

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SAFETY GUIDELINES The electric current in a gel electrophoresis chamber can be dangerous. Always turn off the power supply before removing the lid or touching the gel. Make sure that the counter where the gel is being run is dry. MATERIALS: Per class Bio-Sage Coomassie Blue stain (destains with dH2O) Practice gel loading solution 10X Tris-glycine-SDS buffer stock Vortex mixers Heating block set at 95°C 1000 mL graduated cylinder dH2O 1 L Corning orange capped bottles Plastic wrap White light box Polaroid camera and film A supply of transfer pipettes Labeling tape 1.5 ml microfuge tubes Per every 2 teams Flat metal spatula for separating gels Dual vertical mini gel rig with clamps Power supply Per team Kaleidoscope prestained protein molecular weight markers – aliquot 25 μl per team (BioRad # 161-0324) NOTE: heat briefly at 37°C to dissolve any precipitated SDS before aliquoting.

Flowering plants, one/team 2x Protein loading dye/buffer (≥ 100 ul) Scalpel with sharp blade Precast 15% polyacrylamide gel for SDS PAGE 8 disposable pellet pestles 2 ml 1X Laemmli buffer 1000 l micropipetter and tips 20 l micropipetter and tips Metric ruler Scissors Sharpie marker Small beaker of 10-15 1.5 ml microtubes Microtube rack Plastic dish for transport of gel Plastic staining dish

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

RECIPES 10x Tris-Glycine-SDS Buffer (500 ml should be allowed for each double gel rig; 1x = 25 mM Tris; 192 mM glycine, 0.1 % SDS ) 3.04 g Tris base 14.41 g glycine 1.0 g SDS dH2O to give 100 ml final volume Dilute 100ml in 900ml dH2O for 1x working concentration 10 x Laemmli Buffer 0.25 M Tris, 1.92 M Glycine 1 % SDS in aqueous solution Dilute to 1x working concentration 2x Protein loading dye (10 ml) 1.2 ml 1M Tris HCl pH 8* 4 ml 10% SDS 2 ml 100% glycerol 1 mg bromophenol blue 0.1 ml -mercaptoethanol 2.7 ml dH2O to give 10 ml final volume *Adjust to pH 8 with HCl for prepoured graduated gels; if using discontinuous self poured gels, adjust pH to 6.8.

PROCEDURE Part I. Prep Teams 1 and 2: Set two heating blocks with 1.5 ml tube blocks at 95°C. Gather supplies needed and distribute to all teams. Set up an ice bucket for each team. Teams 3 and 4: Prepare 2 liters of 1x Tris-Glycine-SDS electrophoresis buffer from the laboratory stock. (Note: Two liters is enough for three gel rigs/6 gels.) Verify your calculation with your instructor before diluting. Label the bottle of buffer per cGMP. Part II. Isolating plant proteins 1. Label 6 or 7 1.5 ml microcentrifuge tubes with your team number and the plant tissue type. 2. Use a sharp scalpel to cut the plant tissues to obtain the equivalent of the size of two Tic-Tac breath mints. Be sure to take the following samples: stem, flower, sepals, roots, top leaves, and bottom leaves. If seeds are available in the lab, they can also be used. Place each tissue into its labeled tube.

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Petals

Sepals

BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

3. Grind each tissue with its own pellet pestle for no less than 5 minutes. Do not cross contaminate samples – use a different pestle for each! 4. After each tissue is ground, add 250 μl of 1x Laemmli buffer and grind for another 3 minutes. 5. Incubate each sample for 5 minutes at room temperature. 6. Add 30 μl of 2x protein loading dye to each sample and flick to mix. 7. Heat the team samples for five minutes at 95 C. Spin for 2 minutes to pellet the plant debris and place on ice until ready to load.

Part III. Running and Staining the Gel The instructor will demonstrate how to set up a vertical gel for SDS-PAGE. 1. Prepare the gel by removing the comb and the piece of tape at the bottom. Clip the gel to the vertical electrophoresis chamber with the short glass plate next to the gasket on the top buffer reservoir. Add running buffer to cover the wells in the top reservoir and enough in the bottom reservoir to immerse the slit in the glass plate exposed when the tape was removed. 2. Any debris and bubbles should be removed from each well by using a transfer pipette to gently flush the wells with running buffer. 3. Check the information that accompanied the gels to verify well capacity, as this may vary depending on the gel manufacturer. Load the gel in the following order. (NOTE: loading dye should be diluted 1/10 if it is too thick.) Lane 1. 10 μl 2x protein loading dye Lane 2. 10 μl molecular weight protein markers Lane 3. 10 μl 2x loading dye Lane 4. 20 μl sample – top leaves Lane 5. 20 μl sample – bottom leaves Lane 6. 20 μl sample – sepals Lane 7. 20 μl sample – stem Lane 8. 20 μl sample – flower Lane 9. 20 μl sample – seeds (or 10μl 2x protein loading dye) Lane 10. 10 μl 2x protein loading dye

(NOTE: vertical polyacrylamide gels sometimes run unevenly due to unequal distribution of the heat generated during running. To help prevent „smiling‟ of the samples, no wells should be left empty.) Gel showing „smiling.‟

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

4. Run the gels at 125 volts until the bromophenol blue dye front migrates to the end of the gels, which takes about 45 minutes. Do not let the dye front run off the gel. 5. Add 50 ml Coomassie stain to a small plastic container. 6. When the gel is finished running, turn off the power supply and remove the lid. Gently pry the glass plates apart using a spatula. Submerge the plate, gel-side-down, into the stain to allow it to release from the glass. 7. Place the lid on the container and shake gently for 30 minutes. 8. Pour off the stain and add 100 ml of destaining solution. Shake gently for 30 minutes. Change the destain solution, and continue shaking until the protein bands are visible. The gel can be left in destain overnight or longer. 9. Place the destained gel on a white light box and take a photo. The Polaroid camera should have a yellow filter, and an f-stop setting of 22 - 32. An exposure time of 1/125 second may give good results but this will depend on each light box. For digital photos, use the white light illuminator and the hood from the Polaroid lab camera to obtain the correct distance above the gel.

DATA ANALYSIS Part I. Manual Graphing The proteins will appear as blue bands. The size of the most abundant proteins can be determined by setting up a standard curve using the size of the molecular weight markers and the distance each marker band migrated from the well. Measure the distance (in millimeters) that each band of protein migrated from the bottom of the well to the middle of each band and record in the data table. Measure the distance that the dye front migrated in each lane and record it in the table. Calculate the relative mobility (Rf) for each protein by dividing the distance migrated by the distance the dye front migrated. Set up a data table to include the distance each protein migrated, the distance the dye front migrated at the bottom of each lane, the Rf value for each, and the molecular weight of each protein. Use semi-log graph paper to graph the standard curve by placing the molecular weight of each marker on the Y (ordinate) axis of the graph and the Rf value on the X (abscissa) axis. See a sample data table and sample graph in Table 1 and Figure 1, below. Include your data table and this graph in your notebook as part of your results. (NOTE: the log of the molecular weight size of each standard protein should also be calculated and added to the table for use in Part II.)

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

Table 1. Data Table of SDS PAGE Results Standards & Unknowns

Distance of Migration

Distance Dye Front Migrated in this lane

Rf (X axis)

Molecular Weight in daltons* (Y axis)

log mol. Wt. (for use in Part II)

Myosin (blue) Beta-galactosidase (magenta) BSA (green) Carbonic anhydrase (violet) Soybean trypsin inhibitor (orange) Lysozyme (red) Aprotinin (blue)

Unknown protein 1 Unknown protein 2 Unknown protein 3 *The dye attached to each of the standard proteins causes each to run a little more slowly. These sizes are not the actual size but are corrected for the dye content on each. Kaleidoscope Markers have different molecular weights for each lot – CHECK THE DATA SHEET ENCLOSED WITH THE MARKERS.

Figure 1. Example of a Standard Curve for Protein Molecular Weight Determination

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

Part II. Graphing a Standard Curve using Excel. Graphing with Excel is more accurate than a manually drawn graph. By using the formula for the slope of the line, y = mx + b (slope = m; y intercept = b)

Excel formulas can be used to automatically calculate the size of unknown proteins also run on the same gel. Set up Data Table 1 in an Excel spreadsheet. Use Excel to graph the standard curve by placing the log of the molecular weight of each marker on the Y (ordinate) axis of the graph and the Rf value on the X (abscissa) axis. Use the formula for a line and Excel functions to determine the sizes of the unknown proteins on the gel. Do not round off any calculations; use all six places to the right of the decimal. The function to calculate the log is Log10. For anti-log calculations, select the “Power” function and use 10 for “number” and the log for “power.” Print your data table with the embedded graph and include it in your results. The table and graph must include appropriate labels and legends. A sample Excel data table and graph are given, below. SDS-PAGE Determination of Unknown Protein Size Distance Distance dye front of migrated migration (mm) (mm)

23 29 35.5 43 27 32 36 y=mx+b

45 45 45 45 45 46 45 m=slope

Rf (X axis)

Log Mol. Molecular Wt. (y) Weight

0.51 4.97 0.64 4.83 0.79 4.63 0.96 4.48 0.60 4.866449 0.70 4.755449 0.80 4.644449 -1.11

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ID

94000 MWt Marker " 67000 " 43000 " 30000 73,527 Unknown B 56,944 Unknown C 44,101 Unknown D intercept = b

5.5324491

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

QUESTIONS 1. What were the sizes, based on your graphs, of the major proteins observed? Compare these data with that in other groups to determine if there are any patterns that emerge, i.e., did all plants have the same major protein bands for the same plant structure (e.g., leaves)? 2. Use the Internet to find the kilodalton sizes of some abundant plant proteins, e.g., rubisco (enzyme responsible for fixing carbon). Articles can be found on the class Blackboard website that may help get you started. 3. Explain the action of -mercaptoethanol and SDS in preparation of proteins for SDS PAGE. 4. Explain the difference between native polyacrylamide gel electrophoresis and denaturing PAGE. 5. A protein migrates on SDS-PAGE at the same speed as the 80,000 Dalton molecular weight marker fragment. When the protein is further analyzed using other instrumentation, it is found to have a mass of 65,000 Daltons not including the carbohydrate moiety covalently attached to it. Assuming that the latter is the correct size of the protein, why did it indicate a mass of 80,000 Daltons using SDS-PAGE? 6. IgG contains 2 small and 2 large polypeptide chains. A preparation of IgG was incubated with SDS, heated and electrophoresed using SDS polyacrylamide. One major band near the top of the gel was observed after staining. Explain these results. 7. Compare and contrast the results from your semi-log hand drawn graph and your Excel graph. 8. The size of DNA fragments that have migrated in an agarose gel can also be determined by using a standard graph of the molecular weight marker fragments. Since DNA fragments normally run uniformly across an agarose gel, the Rf is not used, but rather the distance the fragments actually migrated in the gel. Complete the Excel Graphing Practice on the next page. Email your Excel spreadsheets (with the graphs on the spreadsheet for each) to your instructor by the due date given.

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

EXCEL GRAPHING PRACTICE Construct a data table and graph to determine the unknown DNA or Protein bands in the following three examples. Formulas can be viewed by going to TOOLS, OPTIONS, VIEW and clicking FORMULAS.

1. Use the results below to find the sizes of DNA bands that migrated 3.25, 3.55, and 4.55 cm on the gel. (NOTE: For DNA data results, the Rf is not usually used, but rather the distance the fragments actually migrated in the gel.) Mwt band Distance Log bp size (bp) migrated size (cm) 9,416 6,557 4,361 3,000 2,322 2,027 725 570

2.39 2.7 3.12 3.55 3.8 3.99 5.3 5.5

3.97 3.82 3.64 3.48 3.36 3.31 2.88 2.76

2. Determine the size of two unknown DNA bands that migrated 2.45 and 2.03 cm on this gel. Mwt band Distance Log bp size (kb) migrated size (cm) 10 8 6 5 4 3 2.5 2 1.5 1 0.5

1.4 1.52 1.7 1.84 2 2.2 2.4 2.55 2.77 3.1 3.6

4 3.9 3.78 3.7 3.6 3.48 3.4 3.3 3.18 3 2.7

3. Use the following results to determine the size of DNA bands that migrated 4.65, 4.49, 4.75, and 4.82 cm each. Distance of migration (cm)

Log of bp size

Size of fragment (bp)

3.15

3

1000

3.6

2.9

800

4.2 5.03

2.78 2.6

600 400

6.35

2.3

200

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BIOTECHNOLOGY I – PAGE DETERMINATION OF PROTEIN FINGERPRINTS

ANSWERS 1. Data Table for Practice Question 1 Unknown bp size B (antilog Y)

solve for Y x: distance migrated

4,038 3.606225

3.25

Slope of the line (m): Y intercept (b):

3,118 3.493833 1,316 3.119193

3.55 4.55

Y = mx+b

-0.37464 4.823805

Unknown C

2. Data Table for Practice Question 2 -0.57541Slope (m) 4.770114y intercept (b) bp size y=mx+b

y value

2,293

Distance supercoiled plasmid migrated (cm)

3.36036

2.45 Distance relaxed plasmid migrated (cm) 3,999 3.602032 2.03

3. Data Table for Practice Question 3 migration Log of bp size (cm)

bp size Y = mx+b

4.65 2.675908

474

4.49

2.71078

514 slope (m) = -0.21795

4.75 2.654113 4.82 2.638856

451 intercept = 3.689375 435

Sources: Ausubel, F.M. et al. Current Protocols in Molecular Biology, New York. John Wiley & Sons, 1994-2001. http://csm.jmu.edu/biology/courses/bio480_580/mblab/rubiscointro.htm http://www.ingentaconnect.com/content/cabi/ivp/2004/00000040/00000002/art00004 http://sunflower.bio.indiana.edu/~rhangart/courses/b373/lecturenotes/cellwall/cellwall.html http://www.findarticles.com/p/articles/mi_m3741/is_1_53/ai_n8699055 Sambrook, J., Russell, D.W. Molecular Cloning: A Laboratory Manual, 3rd Edition. Cold Spring Harbor, N.Y. Cold Spring Harbor Laboratory Press, 2001. Thiel, T., Bissen, S., Lyons, E. Biotechnology: DNA to Protein; A Laboratory Project. McGraw Hill. 2002.

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