Lab 5_SDS PAGE

7/25/2019 Lab 5_SDS PAGE

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!Barbara T. Nash, Ph.D., Mercy College September 2012

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Introduction to SDS-Polyacrylamide Gel Electrophoresis

Electrophoresisis defined as the transport of charged particles by an electrical field. Whenplaced in an electrical field, any ion will migrate towards the electrode of opposite charge.Because most biological polymers carry a net charge at any pH other than their isoelectric point,

they will migrate at a rate proportional to their charge. The electrophoretic mobility(rate ofmigration) of a molecule through an electrical field will depend on the strength of the field, andon the net charge, size and shape of the molecule.Zonal Electrophoresis, the electrophoresis ofpolymers in a support matrix, is used both as a preparative and analytical technique in the studyof nucleic acids and proteins. Because the most commonly used support matrices are agarose andpolyacrylamide gels, the more familiar name for this technique isgel electrophoresis.

The support matrix in gel electrophoresis is a porous medium that acts as a molecular sieve. Itretards the movement of macromolecules while allowing small molecules to migrate freely.Unlike the sieving process of gel filtration chromatography, in which large molecules areexcluded from the matrix, in gel electrophoresis, all of the migrating molecules enter the gel.Within the gel, larger macromolecules experience a greater degree of retardation and migrate

more slowly than smaller ones. The extent of retardation depends on how closely the gel poresize approximates the size of the migrating molecules. Agarose gels, which form large pores, areused to separate very large macromolecules such as nucleic acids, very large proteins, andprotein complexes. Polyacrylamide, which makes a smaller pore gel, is suitable for theseparation of most proteins, and smaller nucleic acid fragments. Polyacrylamide gels areproduced by the polymerization of acrylamide monomers and the cross-linking of the polymersby the bifunctional agent, bisacrylamide. The greater the concentration of acrylamide (%T), thesmaller the pore size of a gel. Gels are generally prepared as a fixed percentage of acrylamide(e.g. 5%, 10%, 12%) or as a gradient (e.g. 4-15%). Provided one has an idea about the type ofproteins present in a sample mixture, it is possible to choose a gel formulation that will give thebest possible separation.

In order to use gel electrophoresis to determine the molecular mass of a protein, the molecularsieving process must be based on size alone. In order for this to occur, all proteins must have thesame charge density(charge to mass ratio) and the same shape. Treatment of proteins with theanionic detergent, Sodium dodecyl sulfate (SDS), accomplishes this. SDS is an anionic detergentthat denatures proteins and separates the subunits of most multimeric proteins. It is anamphoteric substance that binds proteins via its nonpolar, hydrocarbon tail, while coating theprotein with the negative charge of its ionic sulfate head group. Most proteins bind the sameamount of SDS per gram of protein, forming molecules of identical charge density. These SDS-protein complexes assume the same rod-like (hairpin) shape, the length of which varies with themolecular mass of the protein moiety (Figure 1). Thus, in SDS polyacrylamide gelelectrophoresis (SDS-PAGE), SDS-protein complexes migrate toward the anode (the positive

electrode) with a mobility that is exclusively a function of their molecular masses. Glycoproteinsillustrate the most important exception to this principle. The hydrophilic carbohydrate moietyhinders the binding of SDS and reduces the charge density of the SDS-complexes. Molecularmasses of glycoproteins determined by SDS-PAGE are generally overestimated.

One caution: proteins must be treated with both SDS and a reducing agent with a reactive thiol

group. The two commonly used reagents are "-mercaptoethanol ("-MSH) and dithiothreitol(DTT). Either of these reducing agents breaks disulfide bonds in proteins. If such bonds are left


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intact during the formation of SDS complexes, the polypeptide chains are prevented fromforming the "universal" rod-like structure (Figure 2) and correct molecular masses are notobtained. In addition, in the absence of a reducing agent, the subunits of proteins containingintermolecular disulfide bonds will not separate and multimeric structures will be retained.

Figure 1. Disruption of quaternary protein structure by SDS. The SDS binds tightly to the subunits obscuring theoriginal charge on the protein. In effect, all the SDS-protein complexes that form have the same charge/mass ratio.

Figure 2. The role of "#MSH (or DTT) in the formation of the "universal" rod-like structure of SDS-proteincomplexes.


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SDS gel electrophoresis can be used to determine the subunit molecular mass of a protein.Because the quaternary structure is disrupted, the subunit molecular mass and not the totalmolecular mass of a multimeric protein is determined by this method. A minute amount of asmall, anionic dye, bromophenol blue is added to the protein sample. This dye moves fastest -analogous to using Blue Dextran on a gel filtration column. The gels are normally run until thedye frontapproaches the bottom of the gel. Gels are then stained with Coomassie Blue inaqueous alcohol. This solution both stains and "fixes" (prevents the proteins from moving) thegels. The distance that each protein band and the dye front has migrated is then measured andexpressed relative to the migration distance of bromophenol blue. This value, the relativemobility(R

f), is analogous to the V

e/V

oin gel filtration. It is between 0 and 1 for each protein.

There is a linear relationship between the relative mobility of a protein on an SDS gel and the logofits subunit molecular mass. A standard curve can be constructed (log Mrversus Rf)using

proteins with known subunit molecular masses (Figure 3). The subunit molecular mass of an"unknown" protein can be determined from its R

fby interpolation on the standard curve.

Table 1 Molecular Mass of Standards

Standard Protein RelativeMolecularMass (Mr)

Phosphorylase b 97,000

BSA 66,200Ovalbumin 45,000

Carbonic Anhydrase 31,000Soybean trypsininhibitor

21,500

Lysozyme 14,400

Figure 3. Standard Curve for SDS-PAGE

SDS-PAGE can be used to investigate the subunit composition of a protein. If a single bandappears on the gel and its molecular mass is determined to equal the molecular mass of the nativeprotein, one may conclude that the protein consists of a single subunit (is monomeric). If themolecular mass of the native protein is a multiple of the subunit molecular mass, one maycalculate the number of subunits present in this multimericprotein. However, the presence ofonly one band on an SDS gel does not prove that a protein consists of identical subunits (ishomologous). The protein may consist of heterologoussubunits of identical molecular massesthat differ in amino acid composition. The formation of SDS-complexes will obscure thedifferences in net charge on these subunits. In addition, molecular mass differences betweensubunits that are too small to be resolved on an SDS gel cannot be distinguished by this method.Heterologous subunits of a protein that can be resolved, will appear as separate bands on the geland the molecular mass of each can be determined. SDS gel electrophoresis can be performed onnonhomogeneous preparations if it is possible to locate the position of the protein of interest. Amethod that is routinely used to accomplish this is to transfer the resolved proteins onto amembrane (Western blotting) and to locate the protein with antibodies, a specific probe.

y = -0.9045x + 5.003

R2= 0.9772

4

4.5

5

5.5

0 0.5 1

Rf

LogMr


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The subunit composition of a multimeric protein can be determined by treating protein sampleswith a cross-linking agent before applying them to an SDS gel. The cross-linking agent can reactwith amino acid residues of two polypeptides that are in contact, thereby linking them bycovalent bonds. This reaction is performed under mild conditions so that not all of the subunitsof each molecule of the protein become cross-linked. The product of this reaction is a mixturecontaining molecules representing each possible degree of cross-linking (i.e. monomers, dimers,trimers, etc). Samples of the treated and untreated protein are applied to the gel for comparison.If the protein is composed of homologous subunits, the untreated sample will appear as a singleband on SDS-PAGE and the subunit molecular mass can be estimated. The cross-linked samplewill appear as a series of bands of molecular masses equal to that of multiples of the subunitmolecular mass. This is called a ladder. The molecular mass of the intact multimeric protein canbe estimated from the molecular mass of the band with the lowest Rf. The number of subunits in

the protein equals the molecular mass of the protein divided by the subunit molecular mass(Figure. 4). If the protein is composed of heterologous subunits, deducing subunit compositionby this method can be more complicated but the same principles apply. In either case it is helpfulto have an estimation of the molecular mass of the intact protein by an independent method, suchas gel filtration or equilibrium centrifugation.

+

100,000 ((tetramer)

75,000 (trimer)

50,000 (dimer)

25,000 25,000 (monomer)

Figure. 4. SDS-PAGE of a protein with and without cross-linking. A plus sign indicates treatment with the cross-linking reagent before electrophoresis; a minus sign indicates no treatment.

Conclusion: This protein is a homologous tetramer (one band indicates homologous subunits).Subunit molecular mass = 25,000 daltons

Confirming evidence: Molecular mass estimation from gel filtration = 100,000 daltons


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The two kinds of buffer systems used for SDS-PAGE are continuous and discontinuous systems.In a continuous buffer system, the same buffer (ion composition and pH) is used in the sample,the gel, and the electrode reservoir and the percent agarose is uniform throughout the gel. The gelhas a relatively small pore size. The sample is applied to the gel (the resolving gel) in as small avolume as possible and proteins are separated according to size as they travel through the gel. Ina discontinuous buffer system, the sample buffer, gel buffer, and electrode buffers differ incomposition and the gel is composed of two regions. The upper portion of the gel, thestackinggel, has a relatively large pore size (low percent agarose) and overlays the resolving gel (higherpercent agarose). The sample can be applied in a larger volume than is possible in a continuoussystem because proteins will concentrate orstackwhile they travel through the stacking gel. Thisprocess is indicated by the concentration of the dye front into a straight, sharp line. Becauseproteins enter the resolving gel in a narrow zone before they begin to resolve (destack),discontinuous systems produce better resolution of proteins and are generally preferred.

We will be working with the discontinuous gel system developed by Laemmli, which is the mostcommonly used SDS-PAGE system. The sample and the gel contain Tris-HCl buffer, whereasthe electrode buffer is composed of Tris-Glycine. The gels are also known as Tris-Glycine gels

or Laemmli gels and the sample buffer is known as Laemmli sample buffer.

The protein we will be analyzing has a subunit molecular mass in the 20,000-50,000 range.Resolving power and molecular weight measurements are most accurate in this range on a 12%resolving gel with a 4% stacking gel. However, because we are using a standard containingproteins ranging in molecular mass from 10,000 to 250,000 daltons, we will use a gradient gelinstead of a gel of fixed percentage. In this gel, the concentration of acrylamide ranges from 4%at the top of the gel to 15% at the bottom. This enables the gel to separate proteins over a widemolecular mass range and produces a standard curve that is linear across the entire molecularmass range of the standards. Because the upper portion of the gel contains 4% acrylamide andacts as the stacking gel, there is no need for a separate stacking gel. These gels can be preparedin a relatively short time. However, because unpolymerized acrylamide is a neurotoxin, it is notsuitable for use in an undergraduate laboratory. Precast gels are commercially available and wewill be using these.

References

1. Nelson, D.L.; Cox, M.M.Lehninger Principles of Biochemistry, 5th

ed.; W. H. Freeman: NY,2008; pp 8890.

2. Hawcroft, D.M.Electrophoresis: The Basics; Oxford University Press: NY, 1997..3. Shi, Q.; Jackowski, G. One-dimensional polyacrylamide gel electrophoresis. In Gel

Electrophoresis of Proteins: A Practical Approach, 3rd

ed.; Hames, B.D., Ed.; OxfordUniversity Press: NY, 1998.

4. Farrell, S.O.; Ranallo, R.T.Experiments in Biochemistry: A Hands-on Approach; ThomsonLearning, Inc.: Pacific Grove, CA, 2000; pp. 207242.

5. Boyer, R.Modern Experimental Biochemistry, 3rd

ed.; Benjamin/Cummings: San Francisco,2000; pp 111121, 133135.


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6. Laemmli, U.K. Cleavage of Structural Proteins during the Assembly of the Head ofBacteriophage T4.Nature1970, 227, 680685.


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Biochemistry

Chemistry 354Laboratry Exercise 5

SDS-Polyacrylamide Gel Electrophoresis

Purpose

1. To determine the subunit molecular mass of yeast alcohol dehydrogenase by SDS-PAGE2. To determine the subunit composition of alcohol dehydrogenase

Introduction

As presented in the Introduction to SDS-Polyacrylamide Gel Electrophoresis, the molecular massof the individual subunits of a protein can be determined from their relative mobility (R

f) on an

SDS gel. This is done by interpolation on a standard curve (log Mrversus Rf) constructed by

running proteins of known molecular masses on the same gel as the protein being analyzed The

protein that will be analyzed in this experiment is alcohol dehydrogenase (ADH), prepared frombaker's yeast (Saccharomyces cerevisiae). It is an enzyme used by yeast in the reversiblefermentation reaction, which converts acetaldehyde to ethanol. A highly purified preparation ofthe enzyme will be used in this experiment. Therefore each band that is seen on the gel willrepresent a distinct subunit of the enzyme. By using the subunit molecular mass determined inthis experiment and the total protein molecular mass calculated in Problem Set 5, the subunitcomposition of the enzyme will be determined.

Protein-SDS complexes are prepared by adding gel sample buffer (GSB) to a solution of proteinin a microcentrifuge tube and heating the tubes in a boiling water bath for 5 minutes. Thesamples are then transferred to ice to be cooled. Protein samples must be diluted at least 2-foldwith gel sample buffer, but a higher dilution does no harm. Dry samples may be dissolveddirectly into gel sample buffer. The maximum volume that can be applied to a well on the gel

limits the dilution factor. This volume is 30 L on the Bio-Rad mini gels that we are using.

Approximately 0.11.0 g of protein is needed per band for detection of proteins when gels are

stained with Coomassie blue and completely destained. Application of more than 1.0 g perband is called overloadingand will cause bands to be too broad to accurately determine the Rf.

However if the protein consists of heterologous subunits, multiple bands will be present and

more than 1.0 g of protein will need to be applied in order for there to be 0.11.0 g of proteinper band. More protein may also be needed to see bands if the gel is not completely destained.

The composition of the Bio-Rad Precision Plus Protein Standards is shown in Figure 1. Themolecular masses of these proteins have been confirmed by sequencing, mass spectrometry, and

migration in a Laemmli SDS-PAGE system. Approximately 100 750 g of each proteinstandard was dissolved in 1.0 mL of gel sample buffer and the SDS-complexes were prepared.This sample can be stored at -20C for one year. Before use, the solution must to be allowed toreach room temperature and thoroughly mixed. It is then ready to be applied to the gel. Each

group will be provided with a 30-L sample and 10-L aliquots will be applied to each of twowells on the gel.


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Figure 1. From the Bio-Rad Laboratory Precision Plus Protein Standards instruction manual

Kaleidoscope Prestained standards (Table 1) are prepared by covalently linking dyes to proteinstandards. This makes it possible to monitor the separation of proteins during electrophoresis.The covalent binding of the dyes to the protein is an uncontrolled reaction, and produces lot-to-lot variation in their molecular masses as well as heterogeneity within each sample. Therefore,these standards are not generally used for molecular mass determinations. Prestained standardsare supplied as SDS-complexes. Before use, the sample is heated for 1 min at 40 C to dissolveany solid material and then kept at room temperature until it is applied to the gel. Each group

will be provided with a 30-L sample and 10-L aliquots will be applied to each of two wells onthe gel.

Table 1. The composition of Bio-Rad Kaleidoscope Prestained Standards

Protein Color Typical AverageMolecular Mass (D)

myosin blue 202,000

"#galactosidase magenta 133,000

bovine serum albumin green 71,000

carbonic anhydrase violet 41,800soybean trypsin inhibitor orange 25,000

lysozyme red 16,300

aprotinin blue 7800

Yeast ADH is provided commercially as a lyophilized solid. A concentrated stock solution of theenzyme has been prepared as a 1.0 mg/mL solution in sodium phosphate buffer and stored at -20C. You will dilute the sample 1/10 in gel sample buffer and prepare an SDS-complex of the

protein. Samples of this diluted solution containing 0.25 g, 0.50 g, 1.0 g, and 2.0 g will beapplied to the gel. This makes it possible to determine the optimal amount of ADH needed to getsharp, clearly visible bands.


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ProcedureMaterialsMini-PROTEAN 3 Electrophoresis ModulePower supplyPrecast gel: 4-15% gradient gel in 0.375 M Tris-HCl, pH 8.8Running Buffer: 25 mM Tris base 0.192 M glycine 0.1% SDS, pH 8.5Laemmli Sample Buffer: 62.5 mM Tris-HCl, pH 6.8 containing 2% SDS, 25% glycerol,

0.01% bromophenol blueGel Sample Buffer (GSB): Laemmli Sample Buffer containing 50 mM dithiothreitol

Bio-Safe$Coomassie Blue StainBio-Rad Precision Plus Protein Standards (unstained)Bio-Rad Kaleidoscope Prestained StandardsYeast alcohol dehydrogenase (Sigma) 1.0 mg/mL in 10 mM sodium phosphate buffer, pH 7.5Hot plateWater bath:250 mL beaker containing glass beads, covered with aluminum foilIce bucketMicrocentrifuge

Microcentrifuge tubes250-mL graduated cylinderWash bottle of distilled waterForcepsScalpelSpatulaTimerMarking penDigital micropipets (P-20, P-200) and tipsTransfer pipetWaste beaker

ShakerStaining TrayIdenti-Plugs for 613 mm tubesSix-inch ruler

HazardsThe running buffer contains Tris, glycine, and SDS, which may cause skin and eye irritation.SDS is harmful if swallowed. Laemmli sample buffer contains Tris, glycerol, and bromophenolblue, which may cause skin and eye irritation. Glycerol and bromophenol blue are harmful ifswallowed. The gel sample buffer contains dithiothreitol, which is toxic and harmful ifswallowed. It targets the central nervous system. It is irritating to the eyes, respiratory systemand skin and has an unpleasant odor. Concentrated solutions should be handled in a fume hood.

Bio-Safe$Coomassie stain, Kaleidoscope$Prestained Protein standards, Precision PlusProtein$standards, and Ready gels have no irritating effects if handled according tospecifications. Safety glasses should be worn at all times. Gloves should be worn when workingwith solutions containing DTT and when staining, destaining, and handling the gel


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MethodsSample Preparation

1. Fill an ice bucket with ice.2. Pick up a sample of ADH, GSB, and the molecular mass standards and transfer them to your

microcentrifuge tube rack. The standards are ready to apply to the gel. The ADH samplemust be converted to an SDS-protein complex.

3. Pick up a 250-mL beaker containing glass beads and covered with a sheet of aluminum foil.4. Punch a small hole in the center of the aluminum foil so that a 1-mL microfuge tube can be

suspended from the foil and not fall into the beaker.5. Add water to a height that will cover the bottom of the microcentrifuge tube when it is

inserted into the hole in the aluminum foil.6. Place the 250-mL beaker with the glass beads, the water, and the foil cover on the hot plate

and set the heat setting on about three-quarters of the maximum setting.

7. Dilute 10 L of ADH 10-fold in gel sample buffer.8. Be sure that the microcentrifuge tube containing the ADH sample is shut tightly.9. When water is boiling, place the microcentrifuge tube containing the ADH into the hole in

the foil so that it is suspended in the boiling water bath.10. Boil sample for 5 minutes. Remove the microcentrifuge tube from the bath (you may want to

use a forceps to avoid burning your fingers) and place it on ice to cool.11. When the sample has cooled, transfer it to the microcentrifuge. Be sure that the tube is

balanced against a tube from another group or a blank tube (containing water). Centrifuge forone second (or momentary setting). This is to bring any water droplets that have condensednear the top of the tube back down to the bottom of the tube.

Assembly of the Mini-PROTEAN 3 Module:

Follow the instructions adapted from the Bio-Rad instruction manual (pp.124128). Pay

particular attention to the following modifications listed briefly on page 127:

1. After adding running buffer to the upper chamber, allow the clamping frame to sit on papertowels for a few minutes in order to check for leaks. The paper towel should remain dry.

2. After adding 200 mL of buffer to the tank, tip the electrophoresis module sideways to allowany bubbles that are adhering to the bottom of the gel plate to rise to the surface. Thepresence of large bubbles can insulate the gel from electrical contact.

3. Add an additional 200 mL of buffer to the lower chamber. Buffer contacting the gel platewill help cool the plate and prevent overheating during the run.

Sample Loading

Refer to the Bio-Rad instructions for the sample loading technique.Load the samples according to the following protocol:


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Table 2. Gel Loading Protocol for SDS-PAGE

Well 1 2 3 4 5 6 7 8 9 10

Sample GSB K ADH (0.10 mg/mL) P ADH (0.10 mg/mL) P K GSB

Amt(L) 10 10 2.5 5.0 10 10 20 10 10 10

Amt (g) 0.25 0.50 1.0 2.0

Note: P = Precision Plus Protein Standards, K = Kaleidoscope Prestained Standards, GSB = Gel Sample Buffer

Running the Gel1. Run at 250300 V on the constant voltage setting. The current should start at approximately

6580 mA/gel and drop to approximately 50 mA/gel at the end of the run.2. Run until the dye front reaches the reference line at the bottom of the gel. The run should

take approximately 15 minutes. Try not to let the dye front run off the gel, but it is not adisaster if it does. You will use the lowest molecular mass standard that you can clearly seeas a reference point instead of the dye front because the position of the dye front is not clear

after staining with Bio-Safe$stain.

Staining the Gel1. Add 200 mL of distilled water to the staining tray.2. Use the opening lever to open the plates. Insert the end of the lever with the arrow between

the plates at the four arrows on the cassette. At each of the four corners of the plate, use theteeth on the lever to wedge between the plates of the cassette and crack the plates open usingan upward or downward motion. Once all four corners of the plate are cracked, peel theplates apart. The gel should remain on the larger plate; discard the short, inner plate.

3. Invert the gel over a staining tray, containing 200-mL of distilled water and the gel shouldpeel from the plate into the water. If necessary, pry a small corner of the bottom edge of thegel up from the cassette and remove the gel from the cassette by letting it slide gently into thetray. Touching the gel to the water combined with gravity will help.

4. Label the tray, cover it, and put the tray on the shaker for five minutes.

5. Wash two more times for 5 minutes with 200 mL of distilled water in order to remove theSDS from the gel.

6. Replace the distilled water with 100 mL of staining solution and shake for 30 minutes.7. Discard the staining solution, rinse the gel in distilled water and keep in distilled water to

destain.8. The bands of protein should be clearly visible immediately after removing the stain.

Measurements can be taken immediately, but the gel should be left to destain if time permits.To destain, float foam plugs above the gel to adsorb the stain and place the tray on theshaker.

Measurements and Calculations1. Remove the gel from the distilled water by lifting the basket out of the tray. If bands are

clearly visible, you can take your measurements. If the gel begins to dry while you areworking, it will curl up. Use a wash bottle to wet the gel with distilled water as needed.

2. Decide which lane of the four containing ADH is the best one to use for measurement. Selectthe lane containing the sharpest band that is clearly visible. Select the lane containing thePrecision Plus standards that is closest to the lane containing the ADH sample that you haveselected for measurement.


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3. Measure the migration distance for each protein band by measuring the distance from thebottom of the well to each band of protein. Do this for each standard protein in the lane thatyou have chosen and for ADH.

4. Calculate the Rffor each band by dividing its migration distance by the migration distance of

the fixed reference point you have chosen. Although this reference point was traditionally thedye front, the staining method that we are using changes the appearance of the dye front froma sharp blue line to a broad streak. It therefore cannot be used as a reference point and youshould use a standard instead. Use the lowest molecular mass standard that is clearly visible.The program will recognize its migration distance as the maximum migration distance or"Max". Note that the Rf of your reference band will be 1.00. Be sure you understand howmany significant figures to include in these measurements before you proceed. This value islimited by the accuracy of your measurements of migration distance, not by the molecularmass of the standards. The molecular masses of the standards are known to the nearestdalton. For instance, standard #1, of molecular mass 250 kD is actually known to be 250,000daltons.

5. Record this data in the graphing software file that you choose (explained below). The tablewill look similar to the one shown below.

Table 3. Data for the SDS-PAGE Standard CurveStandard Migration

Distance (mm)Rf Molecular

Mass(kD)Log Mr

1 250.000 2.3982 150.000 2.176

3 100.000 2.0004 75.000 1.875

5 50.000 1.699

6 37.000 1.5687 25.000 1.3988 20.000 1.301

9 15.000 1.17610 10.000 1.000

ADH

Three Microsoft Excel templates have been created for this experiment. They are located inthe Biochemistry Graph Template folder on the computers in room 315. See page 5 of thismanual for instructions on where to find these files. Depending on how the gel runs, you may

be able to distinguish 8, 9, or 10 standards. Because of a software problem, the program can'tfunction properly in a situation where one of the variables is derived (Rf) and the number ofdata points varies. Therefore, three distinct files have been programmed that contain 8, 9, or10 standards. Select the file that is appropriate for your data. The molecular mass of eachstandard has been entered into the program and the values of log Mrhave been calculated.All you need to do is enter your migration distances and the program will calculate the Rfbydividing each migration distance by the maximum migration distance (that of the lowestmolecular mass standard).


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6. The Excel program will create a standard curve by plotting log Mrversus Rffor the standard

proteins and draw the best straight line through the data points by using regression analysis.It will report the equation for the line and its R

2value. A good straight line should have an R

2

value of 0.970 - 0.999. This means that the data points fit on the line well. Note that the samedefect in the software described above causes the program to create a default (bogus) graph

before you enter your data. The equation for this line is y = -0.15x + 2.48. Ignore it. Thecorrect graph and equation will be displayed when your data is entered.

7. Use the standard curve to estimate the subunit molecular mass for ADH. This is done bysolving the regression equation for x = the Rffor ADH and getting the antilog of your yvalue. The program will do this for you and the value for the subunit molecular mass of ADHshould appear in the data table as soon as you enter your measurements.

8. Calculate the accepted value for the subunit molecular mass of ADH from the informationavailable on the ExPASy web site (4). Enter "alcohol dehydrogenase baker's yeast" into thesearch box. You will get 15 hits, five of which (ADH1-ADH5) are the isozymes of ADHfrom Sacchromyces cerevisiae. Select ADH1, the predominant form in metabolically active

baker's yeast. The subunit molecular mass that is reported here has been calculated from theamino acid sequence. This is the subunit molecular mass of the unprocessed apoprotein.Subtract the molecular mass of the N-terminal extension in order to obtain the molecularmass of the processed apoprotein. Note that the protein is modified in two ways: it iscovalently modified and contains zinc. Add the contribution to the subunit molecular weightfrom the covalently bound group and the zinc atoms to obtain the true subunit molecularmass of ADH. Ignore the phosphoryl groups in your calculation. These are added in responseto a cell signal under specific conditions and are not present on the protein that was used inthis lab.

9. Use the accepted value of the subunit molecular mass to calculate the percent error in your

estimation.

Photographing the Gel (Gel Doc-it System)A. Set-up

1. Turn the power on and place a flash drive into the USB drive.

2. Place the Coomassie Blue filter in the filter tray.

3. Select the transilluminator as the light source (Trans) and wavelength (365 nm).

4. Roll out the transilluminator from the dark room and place the UV to white lightconverter plate on the transilluminator.

5. While wearing gloves, center the gel on the converter plate.

6. Roll the transilluminator back into the dark room.

7. Turn on the UV lamp and shut the door. You can see the gel through the viewport


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B. Photographing the gelNote: Use the touch-pen to operate software controls. The pen is kept in a holder, attachedto the right side of the instrument.

1. Select LIVEfrom the touch screen to see the image of the gel.

2. Rotate the aperture (upper ring on the camera) and adjust the exposure time (+ and keyson the touch screen) to optimize the brightness of the image.

3. Rotate the zoom lens adjustment (middle ring on the camera) so that the gel fills theframe and the image is as big as possible.

4. Rotate the focus adjustment (lower ring on the camera) so that the image is in clear focus.

Note: While the program is in theLIVE mode(LIVEbutton is yellow), the image willautomatically update after each adjustment. Wait for the screen to refresh before makingadditional changes.

5. When you are ready to take a photograph, change the wavelength selection to 320 nm.

6. Save the imagea. Select TIME STAMPto add a code number and date stamp to the photo.a. Select SNAPto capture the image.c. Select SAVEto save the image to the flash drive. The software has been pre-

programmed to save to the flash drive as a jpg file. Other options can be selectedfrom the Preferences menu.

d. When the image has been saved, the SAVEbutton will revert from yellow to white,indicating that the flash drive may be removed from the USB port.

7. Insert the flash drive into a computer and drag the file to the Photoshop icon (the feather)in the dock to open the file in Adobe Photoshop.

8. From the Imagemenu, select Brightness and Contrast. Decrease the brightness andincrease the contrast of the image.

9. From the Imagemenu, select Size. Adjust the width of the image to match that of the gel(3.5 inches).

10. Use the selection tool on the left toolbar to create a tight border around the gel andexclude the staining tray from the image. From the Imagemenu, select Cropto completethis task.

11. Save the edited image on your flash drive and print copies of the photograph for your labreport.

12. Discard the gel in the trash and wash the electrophoresis box, clamping frame, electrodecore, and buffer dam. Rinse the equipment in distilled water and leave it to drain onclean paper towels.


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Report

Your lab report should include the following:

Figures and Tables1. Table 1. Data for the SDS-PAGE Standard Curve2. Figure 1. Standard Curve for the Subunit Molecular Mass Determination of ADH

Report1. Your estimation of the subunit molecular mass of ADH2. The accepted value of the subunit molecular mass of ADH3. Your percent error4. Calculate the total number of subunits in ADH.

a. Hint: There is information in Problem Set 4 that will help you with this calculation.b. If the error in this experiment is large, the experimental value of the subunit molecular

mass will give erroneous results in this calculation. If this occurs, use the accepted valueof the ADH subunit that you obtained from the ExPASy site.

5 What is the subunit composition of ADH? Use the appropriate term and explain how youdetermined this.

6. What is the optimal amount (in g) of ADH to run on this gel?

References

1. Molecular Weight Determination by SDS-PAGE; tech note 3133; Bio-Rad Laboratories:Hercules, CA, 2004.

2. Using Precision Plus Protein Standards to Determine Molecular Weight; tech note 3144;

Bio-Rad Laboratories: Hercules, CA, 2004.

3. Dayhoff, M.O.Atlas of Protein Sequence and Structure; The National Biomedical ResearchFoundation: Silver Spring, MD, 1972; Vol 5, pp D82.

4. ExPASy Protein knowledgebase: http://www.uniprot.org

5. Leskovac, V.; Trivic, S; Pericin, D. The three zinc-containing alcohol dehydrogenases frombaker's yeast, Saccharomyces cerevisiae.FEMS Yeast Research2002,2, 481494.


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Instructions for the Set-Up and Assembly of the Mini-Protean 3 Electrophoresis System

General Information

IntroductionThe Mini-Protean 3 cell runs both hand cast gels and precast gels (Ready gels or TGX gels)interchangeably. The Mini-Protean 3 system includes a casting stand and glass plates withpermanently bonded gel spacers that simplify hand casting and eliminate leaking during casting.The cell can run one or two gels, and the mini tank is compatible with other Bio-Rad electrodemodules for tank blotting, 2-D electrophoresis, and electro-elution.

Components

Buffer Dam The molded, one-piece buffer dam is used when running only one gel.Electrode Assembly The Electrode Assembly holds the Gel Cassette Sandwich. It houses

the sealing gasket, the upper and lower electrodes, and the connectingbanana plugs. The anode (lower electrode) banana plug is identified

with a red marker and the cathode (upper electrode) banana plug witha black marker.

Clamping Frame The Clamping Frame holds the Electrode Assembly and Gel CassetteSandwich in place. Its pressure plates and closure cams seal the GelCassette Sandwich against U-shaped gaskets on the ElectrodeAssembly to form the inner buffer chamber.

Inner Chamber The Electrode Assembly, two Gel Cassette Sandwiches or one gelcassette sandwich and a buffer dam, and the Clamping Frame form theInner Chamber.

Mini Tank and Lid The Mini Tank and Lid combine to fully enclose the inner chamberduring electrophoresis. The lid cannot be removed without disrupting

the electrical circuit. The Mini Tank and Lid are also compatible withother Bio-Rad electrode modules for blotting, first dimension 2-D, andelectro-elution.

Set up and Basic Operation

A. TGX Gel Cassette PreparationNote:The Mini-Protean 3 cell is guaranteed for use only with Bio-Rads Ready Gel or TGXprecast gels.

1. Remove the TGX gel from the storage pouch by gasping the pouch firmly and tearing atone of the notches.

2. Remove the tape at the bottom of the cassette by peeling it off the bottom edge of thecassette.

3. Remove the comb by pushing it up grasping the comb from the notch at the center andlift the comb straight up. Rinse the wells thoroughly with either distilled water (for apractice set-up) or running buffer (for a run).

4. Repeat for a second gel.

Note: If only one gel is to be run, use the mini cell buffer dam to close the inner chamber.


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B. Mini-Protean 3 Electrophoresis Module Assembly1. Remove the Clamping Frame from the Mini-Tank. Rotate the cams of the Casting Frame

outward to release the Electrode Assembly from the Clamping Frame (Figure 5a).2. Place a TGX gel cassette into the slots at the bottom of one side of the Electrode

Assembly. Be sure that the short plate of the TGX gel cassette faces inward toward thenotches of the U-shaped gaskets (Figure 5b).

3. Place the plastic buffer dam on the other side of the Electrode Assembly. Position thebuffer dam so that the writing on the plastic faces inwardtowards the gaskets. If you canread the writing the buffer dam is facing the wrong way.

4. Press the gel cassette and the buffer dam up against the green gaskets. The cassette,buffer dam, and the Electrode Assembly fit together to form the inner chamber of theelectrophoresis unit.

5. Slide the Electrode Assembly, with the plates into the Clamping Frame (Figure 5c).6. Press downon the Electrode Assembly while closing the two cam levers of the Clamping

Frame. This closes the Inner Chamber and ensures a proper seal of the short plate againstthe notch on the U-shaped gasket (Figure 5d). The short plate must align with the notchin the gasket.


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Note:Gently pressing the top of the Electrode Assembly forces the top of the short glassplate on the gel cassette to seat against the rubber gasket properly. If you do not pushdown, the upper buffer may leak, creating a short circuit.

7. Lower the Inner Chamber Assembly and Clamping Frame into the Mini Tank. Fill theinner chamber with approximately 125 mL of running buffer, so that the buffer reaches alevel between the top of the short plate and the top of the long plate of the gel.

Note:Do notoverfill the inner chamber so that the buffer spills over the top of the longplate. Overfilling may cause siphoning of the buffer, resulting in buffer loss andinterruption of electrophoresis due to a short circuit.

8. Add 200 mL of running buffer to the Mini Tank (lower buffer chamber), perform a leaktest, remove air bubbles from the bottom edge of the gel cassette if necessary, and add anadditional 200 mL of running buffer to the Mini Tank.

C. Sample Loading

Note:The sample wells are outlined and labeled for easy sample loading and sampleidentification.

1. Load samples using an L-10 digital micropipette.2. The Sample Loading Guide assists in aligning the pipette tip and the wells. If using the

Sample Loading Guide, place it between the gel cassette and buffer dam in the ElectrodeAssembly. Insert the pipette tip into the slots of the guide and fill the correspondingwells.Note:Load samples slowly to allow them to settle evenly on the bottom of the well. Becareful not to puncture the bottom of the well with the pipette tip.

Figure 6. Using the Sample Loading Guide


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D. Gel Electrophoresis1. Remove the Sample Loading Guide and place the Lid on the Mini Tank. Make sure to

align the color coded banana plugs and jacks. Matching the jacks on the lid with thebanana plugs on the Electrode Assembly makes the correct orientation. A stop on the lidprevents incorrect orientation.

2. Insert the electrical leads into a suitable power supply with the proper polarity. Insert thered plug into the red slot in the power supply and insert the black plug into the black slot.If this orientation is reversed, the samples will migrate out of the wells into the buffer ofthe inner chamber when electrophoresis begins.

3. Apply power to the Mini-Protean 3 cell and begin electrophoresis; 200 volts at constantvoltage is recommended for SDS-PAGE. Run time is approximately 15 minutes for TGXgels.

E. Gel Removal1. After electrophoresis is complete, turn off the power supply and disconnect the electrical

leads.2. Remove the tank lid and carefully lift out the Inner Chamber Assembly. Pour the buffer

from the Inner Chamber out into the tank.

Note:Always pour off the buffer before opening the cams to avoid spilling the buffer.

3. Open the cams of the Clamping Frame. Pull the Electrode Assembly out of the ClampingFrame and remove the gel cassette.

4. Use the opening level to pry open the plates of the gel cassette. Insert the end of the leverwith the arrow between the plates at the four arrows on the cassette. At each of the fourcorners of the plate, use the teeth on the lever to wedge between the plates of the cassetteand crack the plates open using an upward or downward motion. Once all four corners ofthe plate are cracked, peel the plates apart. The gel should remain on the larger plate;discard the short, inner plate.

5. Invert the gel over a staining tray, containing 200-mL of distilled water and the gelshould peel from the plate into the water. If necessary, pry a small corner of the bottomedge of the gel up from the cassette and remove the gel from the cassette by letting itslide gently into the tray. Touching the gel to the water combined with gravity will help.

6. Rinse the Mini-Protean 3 cell electrode Assembly, Clamping Frame, Mini Tank, bufferdam, and gel loading guide in laboratory soap solution. Rinse thoroughly with tap water,and rinse again in distilled water. Leave all equipment on paper towels to air dry.


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Problem Set 5: SDS PAGE and Subunit Structure of Proteins

1. The enzyme, glutathione peroxidase, elutes from a calibrated Sephadex G-100 column at aposition corresponding to a molecular mass of 88,000. When this enzyme is run on SDS gelelectrophoresis a single band with a molecular massof 22,000 is observed. What conclusionscan be drawn about the structure of glutathione peroxidase?

2. The subunit composition of a multimeric protein can be deduced using SDS-PAGE inconjunction with a protein cross-linking agent. This bifunctional reagent can react withamino acid residues of two polypeptides that are in contact, thereby linking them by covalentbonds. After limited treatment with the reagent, so that some but not all subunits becomecross-linked to their neighbors, the protein is subjected to SDS-PAGE and the molecularmasses of the resulting bands are estimated. The results of such experiments on two differentproteins are shown in the figure below. What is the most likely subunit structure of eachprotein?

Protein A Protein B

+ +

85,000

60,000

50,000

40,00035,000

20,000

Figure 1. SDS-PAGE of two proteins with and without cross-linking. A plus sign indicates treatment with the cross-linking reagent before electrophoresis; a minus sign indicates no treatment


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3. You have a protein that is a heterologous trimer of two subunits of molecular mass, 20,000and one subunit of molecular mass, 15,000. On the figures below, draw the results you wouldexpect to obtain if the protein is run on SDS-PAGE before () and after (+) limited treatmentwith a crosslinking reagent. Show the protein bands you would expect to see and indicatetheir molecular masses.

+

Lab 5_SDS PAGE

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