edvo-kit - 154.68.126.6

EVT 011285AM

The Biotechnology Education Company®

®

The Biotechnology Education Company ® • 1-800-EDVOTEK • www.edvotek.com

All components are intended for educational research only.They are not to be used for diagnostic or drug purposes, noradministered to or consumed by humans or animals.

153EDVO-Kit #

Determination of ProteinMolecular Weight

Storage:Some components require freezer storage.

See page 3 for details.

EXPERIMENT OBJECTIVES:

The objective of this experiment is to develop anunderstanding of protein structure and to

determine the molecular weight of unknownprestained proteins by denaturing

SDS-polyacrylamide gel electrophoresis.

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EDVO-Kit # 153 Determination of Protein Molecular Weight

EVT011285AM

Table of Contents

Page

Experiment Components 3

Experiment Requirements 3

Background Information

Determination of Protein Molecular Weight 4

Experiment Procedures

Experiment Overview 9

Protein Denaturation 10

Electrophoresis of Proteins 11

Staining the Gel 14

Determination of Molecular Weights 16

Study Questions 18

Instructor's Guidelines

Notes to the Instructor 19

Pre-Lab Preparations 20

Idealized Schematic of Results 22

Study Questions and Answers 23

Material Safety Data Sheets 25

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EDVOTEK - The Biotechnology Education Company ®1-800-EDVOTEK • www.edvotek.com

24-hour FAX: (301) 340-0582 • email: [email protected]


EVT011285AM

Component StorageA Standard Prestained Protein Marker Freezer with desiccantB Unknown Prestained Protein 1 Freezer with desiccantC Unknown Prestained Protein 2 Freezer with desiccantD Unknown Prestained Protein 3 Freezer with desiccant

Tris-glycine-SDS buffer (10x) Room temperatureProtein InstaStain® sheets Room temperatureProtein Plus™ stain (10x ) Room temperaturePractice gel loading solution Room temperatureSemi-log graph paper templateTransfer pipets

LyphoProtein™ samples are protein samples which are denatured, lyophilizedand ready for electrophoresis after rehydration and heating.

None of the components have been prepared from human sources.

Experiment Components

• MV10 vertical electrophoresis apparatus• D.C. power supply• Three 12% pre-cast SDS polyacrylamide gels (Cat. #651)• Automatic micropipet and tips

(Cat. #638, Fine Tip Micropipet Tips recommended)• Metric rulers• Hot plate or burner• 500ml graduated cylinder• Beakers• Methanol (150ml)• Glycerol (15ml)• Distilled or deionized water• Glacial acetic acid (80ml)• Glass staining tray/dish (optional)• Aluminum foil or microtest tube holder• Scissors• Plastic wrap• Spatula or gel spacer• 500 gram weight• White light box• Small plastic tray or weigh boat• Photodocumentaion system (optional)

RequirementsAll components areintended foreducational researchonly. They are not tobe used fordiagnostic or drugpurposes, noradministered to orconsumed byhumans or animals.

LyphoProtein andProtein Plus aretrademarks ofEDVOTEK, Inc.

Protein InstaStain,EDVOTEK, and TheBiotechnologyEducation Companyare registeredtrademarks ofEDVOTEK, Inc.

There is enough of eachsample for six (6) groups

sharing threepolyacrylamide gels.

Upon receipt,freeze

Components A-D.


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Duplication of this document, in conjunction with use of accompanying reagents, is permitted for classroom/laboratory use only. This document, or any part, may not be reproduced or distributed for any other purposewithout the written consent of EDVOTEK, Inc. Copyright © 1997, 1998, 1999, 2000, 2005 EDVOTEK, Inc., all rightsreserved. EVT011285AM

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Determination of Protein Molecular Weight

Proteins are a highly diversified class of biomolecules. Differences in theirchemical properties, such as charge, functional groups, shape, size andsolubility enable them to perform many biological functions. Thesefunctions include enzyme catalysis, metabolic regulation, binding andtransport of small molecules, gene regulation, immunological defenseand cell structure. Determination of the molecular weight of a protein isof fundamental importance to its biochemical characterization. If theamino acid composition or sequence is known, the exact molecularweight of a polypeptide can be calculated. This assumes that theprotein does not contain any “non-amino acid” chemical groups (heme,zinc, covalently bonded carbohydrate, etc.) or that the amount of thesegroups, if present, is already known. SDS gel electrophoresis is commonlyused to obtain reliable molecular weight estimates for denaturedpolypeptides. Other techniques for the determination of very accuratemolecular weights include analytical ultracentrifugation and light scatter-ing. However, these methods require large amounts of highly purifiedproteins and costly, sophisticated equipment.

A protein can have a net negative or net positive charge, depending onits amino acid composition and the pH. At certain pH values of solutions,the molecule can be electrically neutral, i.e. negative and positivecharges are balanced. In this case, the protein is isoelectric. In thepresence of an electrical field, proteins with net charges will migratetowards the electrodes of opposite charge.

Proteins exhibit different three-dimensional shapes and folding patternswhich are determined by their amino acid sequences and intracellularprocessing. The precise three-dimensional configuration of a protein iscritical to its function. The shapes these molecules can have are spheri-cal, elliptical or rod-like. The molecular weight is a function of the numberand type of amino acids in the polypeptide chain. Proteins can consist ofa single polypeptide or several polypeptides specifically associated witheach other. Proteins that are in their normal, biologically active forms arecalled native.

The physical-chemical properties of proteins affect the way they migrateduring gel electrophoresis. Gels used in electrophoresis (e.g. agarose,polyacrylamide) consist of microscopic pores of a defined size range thatact as a molecular sieve. Only molecules with net charge will migratethrough the gel when it is in an electric field. Small molecules passthrough the pores more easily than large ones. Molecules having morecharge than others of the same shape and size will migrate faster.Molecules of the same mass and charge can have different shapes. Insuch cases, those with more compact shape (sphere-like) will migratethrough the gel more rapidly than those with an elongated shape, like arod. In summary, the charge, size and shape of a native protein all affectits electrophoretic migration rates. Electrophoresis of native proteins isuseful in the clinical and immunological analysis of complex biologicalsamples, such as serum, but is not reliable to estimate molecular weights.



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POLYACRYLAMIDE GEL ELECTROPHORESIS

Polyacrylamide gels are formed by mixing the monomer, acrylamide, thecross-linking agent, methylenebisacrylamide, and a free radical genera-

tor, ammonium persulfate, in aqueous buffer.Free radical polymerization of the acrylamideoccurs. At various points the acrylamidepolymers are bridged to each other (as shownin Figure 1). The pore size in polyacrylamidegels is controlled by the gel concentration andthe degree of polymer cross-linking. Theelectrophoretic mobility of the proteins isaffected by the gel concentration. Higherpercentage gels are more suitable for theseparation of smaller polypeptides. Polyacryla-mide gels can also be prepared to have agradient of gel concentrations. Typically thetop of the gel (under the sample wells) has aconcentration of 5%, increasing linearly to 20%at the bottom. Gradient gels can be useful inseparating protein mixtures that cover a largerange of molecular weights. Gels of homoge-neous concentration (such as those used in thisexperiment) are better for achieving widerseparations of proteins that occupy narrowranges of molecular weights.

It should be noted that acrylamide is a neuro-toxin and can be absorbed through the skin.However, in the polymerized polyacrylamide

form it is non-toxic. The polymerization process is inhibited by oxygen.Consequently, polyacrylamide gels are most often prepared betweenglass plates separated by strips called spacers. As the liquid acrylamidemixture is poured between the plates, air is displaced and polymerizationproceeds more rapidly.

Sodium dodecylsulfate (SDS) is a detergent which consists of a hydrocar-bon chain bonded to a highly negatively chargedsulfate group (as shown in Figure 2).

SDS binds strongly to most proteins and causes them tounfold to a random, rod-like chains. No covalentbonds are broken in this process. Therefore, the aminoacid composition and sequence remains the same.Since its specific three-dimensional shape is abolished,the protein no longer possesses biological activity.Proteins that have lost their specific folding patternsand biological activity but have their intact polypep-tide chains are called denatured. Proteins which

Figure 2: The chemical structure of sodiumdodecylsulfate (SDS).

Figure 1:Schematic of polyacrylamide polymer formation.


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contain several polypeptide chains that are associated only by non-covalent forces will be dissociated by SDS into separate, denaturedpolypeptide chains. Proteins can contain covalent crosslinks known asdisulfide bonds. These bonds are formed between two cysteine aminoacid residues that can be located in the same or different polypeptidechains. High concentrations of reducing agents, such as b-mercaptoethanol, can break disulfide bonds. This allows SDS to com-

pletely dissociate and denature theprotein. Proteins that retain theirdisulfide links bind less SDS, causinganomalous migration. Figure 3 illus-trates a protein containing two differ-ently sized polypeptide chains that arecross-linked by a disulfide bond. Thechains are also associated by non-covalent forces. The circles representthe native structure.

Certain membrane proteins form SDScomplexes that do not contain theusual ratio of detergent, causinganomalous migration rates. Proteinsthat are highly glycosylated alsoexhibit anomalous behavior, particu-larly if the carbohydrate units containcharged groups. It should be notedthat SDS does not interact withpolysaccharides and nucleic acids.

In most cases, SDS binds to proteins in aconstant ratio of 1.4 grams of SDS pergram of protein. On average, thenumber of bound SDS molecules is halfthe number of amino acid residues in

the polypeptide. The amount of negative charge of the SDS is muchmore than the negative and positive charges of the amino acid residues.The large quantity of bound SDS efficiently masks the intrinsic changes inthe protein. Consequently, SDS denatured proteins are net negative andsince the binding of the detergent is proportional to the mass of theprotein, the charge to mass ratio is constant. The shape of SDS denaturedproteins are all rod-like. The linear size of the rod-like chains is the physicaldifference between SDS denatured proteins. The larger the molecularweight of the polypeptide the longer the rod-like chain. The electro-phoresis gel pores distinguish these size differences.

During SDS electrophoresis, the proteins migrate through the gel towardsthe positive electrode at a rate that is inversely proportional to theirmolecular weight. In other words, the smaller the denatured polypeptide,

Figure 3:Protein Denaturationin the presence of2-mercaptoethanol.



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the faster it migrates. The molecular weight of an unknown polypeptide isobtained by the comparison of its position after electrophoresis to thepositions of standard SDS denatured proteins. The molecular weights ofthe standard proteins have been previously determined. After proteinsare visualized by staining and destaining, their migration distance ismeasured. The log10 of the molecular weights of the standard proteins areplotted versus their migration distance. Taking the logarithm Rf allows thedata to be plotted as a straight line. The molecular weight of unknownsare then easily calculated from the standard curve.



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PROTEIN SAMPLES FOR THIS EXPERIMENT

The standard molecular weight markers (Lanes 1 and 6) are a mixture ofproteins that give the following denatured molecular weights: prestained94,000; 67,000; 43,000; 30,000; 20,100; and 14,400. The denatured valueshave been rounded off for convenience in graphical analysis.

The protein samples have been denatured with the anionic detergentsodium dodecyl sulfate (SDS). Under the experimental conditions, theproteins will have a mobility in the gel that is inversely proportional to thelogarithm of their molecular weights. Proteins of known molecular weightswill be separated by electrophoresis in parallel and will be used to esti-mate the molecular weights of the unknown protein samples by graphicalanalysis. All protein samples contain buffer, SDS, b-mercaptoethanol asthe reducing agent for disulfide bonds, glycerol to create density greaterthan that of the electrode buffer and the negatively charged trackingdye bromophenol blue. The tracking dye will migrate ahead of thesmallest proteins in these samples towards the positive bottom electrode.The molecular weight estimates obtained from SDS polyacrylamide gelelectrophoresis are of denatured proteins. Since proteins often consist ofmultiple subunits (polypeptide chains), subunit molecular weight informa-tion will be obtained. The molecular weights of the proteins in their nativestates will be provided so that the number of subunits can be determined.The protein samples provided have been purified to approximately 80-90% purity by salt fractionation and column chromatography procedures.Minor protein bands may appear which may be due to aggregation orcontamination.

Since the proteins are prestained, the individual bands will be visibleduring electrophoresis. After electrophoresis, preliminary measurementscan be made without removing the gel from the plastic cassette.

The prestained proteins can be made more visible by placing the gel instaining solution. The proteins are usually precipitated and in the gelmatrix during the staining procedure by a process called fixation. Fixationis necessary to prevent protein diffusion, which causes blurry bands andreduced intensity. Fixatives often include acetic acid and methanol.There are several staining methods. Protein InstaStain® is a new state-of-the-art, proprietary patented staining method available exclusively fromEDVOTEK®. Protein Plus™ stain, also formulated by EDVOTEK®, is designedto give rapid and sensitive staining by the traditional liquid stainingmethod without the use of corrosive chemicals. You may wish to com-pare the different staining methods.



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Wear glovesand safetygoggles

Experiment Overview

EXPERIMENT OBJECTIVE:

The objective of this experiment is to develop a basic understanding ofprotein structure and to determine the molecular weight of unknownprestained proteins by denaturing SDS-polyacrylamide gel electrophoresis.

LABORATORY SAFETY

1. Gloves and goggles must be worn at all times.

2. Unpolymerized acrylamide is a neurotoxin andshould be handled with extreme caution in a fumehood.

3. Use a pipet pump to measure polyacrylamide gelcomponents. Polymerized acrylamide, such asprecast gels, are safe but should still be handledwith gloves.


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Protein Denaturation

The samples are denatured proteins which tend to formsuper-molecular aggregates and insoluble particulates.Heating disrupts metastable aggregates of denaturedproteins.

1. Bring a beaker of water, covered with aluminum foil, to aboil. Remove from heat.

2. Make sure the sample tubes A through E are tightlycapped and well labeled. The bottom of the tubesshould be pushed through the foil and immersed in theboiling water for 3 to 4 minutes. The tubes should bekept suspended by the foil.

3. Proceed to loading the gel while the samples are stillwarm.

Quick Reference:

The heating (Steps 1-2) disruptsaggregates of denatured proteins.Denatured proteins tend to formsuper-molecular aggregates andinsoluble particulates.




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Electrophoresis of Proteins

PREPARING THE POLYACRYLAMIDEGEL FOR ELECTROPHORESIS

Precast Polyacrylamide Gels:

Precast polyacrylamide gels will vary slightly in design. Procedures for theiruse will be similar.

1. Open the pouch containing the gel cassette with scissors. Removethe cassette and place it on the bench top with the front facing up.

Note: The front plate is smaller (shorter) than the back plate.

2. Some cassettes will have tape at the bottom of the front plate.Remove all of the tape to expose the bottom of the gel to allowelectrical contact.

3. Insert the Gel Cassette into the electrophoresis chamber.

4. Remove the comb by placing your thumbs on the ridges and pushing(pressing) upwards, carefully and slowly.

Wear glovesand safety goggles

PROPER ORIENTATION OF THE GEL IN THEELECTROPHORESIS UNIT

1. Place the gel cassette in the electrophoresis unitin the proper orientation. Protein samples will notseparate in the gel if the cassette is not orientedcorrectly. Follow the directions accompanyingthe specific apparatus.

2. Add the diluted buffer into the chamber. Thesample wells and the back plate of the gelcassette should be submerged under buffer.

3. Rinse each well by squirting electrophoresis bufferinto the wells using a transfer pipet.

The gel is now ready for practice gel loading and/or samples.

The figure below showsa polyacrylamide gelcassette in theEDVOTEK® VerticalElectrophoresisApparatus, Model#MV10.

Negative ElectrodeBlack Dot (-)

Buffer Level

Sample Well

Long Cassette Plate

Polyacrylamide Gel

Viewing Level(cut out area on

front support panel)

Platinum wire

Short Cassette Plate

Gel Cassette Clip

Micropipet withFine tip


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PRACTICE GEL LOADING

EDVOTEK® Cat. #638, Fine Tip Micropipet Tips are recommended forloading samples into polyacrylamide gels. A regular microtip maydamage the cassette and result in the loss of protein samples.

1. Place a fresh fine tip on the micropipet. Aspirate 20 µl of practice gelloading solution.

2. Place the lower portion of the fine pipet tip between the two glassplates, below the surface of the electrode buffer, directly over asample well. The tip should be at an angle pointed towards the well.The tip should be partially against the back plate of the gel cassettebut the tip opening should be over the sample well, as illustrated inthe figure on page 11.

Do not try to jam the pipet tip in between the plates of the gelcassette.

4. Eject all the sample by steadily pressing down on the plunger of theautomatic pipet.

Do not release the plunger before all the sample is ejected. Prema-ture release of the plunger will cause buffer to mix with sample in themicropipet tip. Release the pipet plunger after the sample has beendelivered and the pipet tip is out of the buffer.

5. Before loading protein samples for the actual experiment, thepractice gel loading solution must be removed from the samplewells.

Do this by filling a transfer pipet with buffer and squirting a stream intothe sample wells. This will displace the practice gel loading solution,which will be diluted into the buffer and will not interfere with theexperiment.

EDVOTEK® Cat. #638,Fine Tip Micropipet Tipsare recommended forloading samples intopolyacrylamide gels.A regular microtipmay damage thecassette and result inthe loss of proteinsamples.

READ ME!




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LOADING PROTEIN SAMPLES

Change fine pipet tips between loading each sample. Make sure thewells are cleared of all practice loading solution by gently squirtingelectrophoresis buffer into the wells with a transfer pipet.

Two groups will share each gel. The prestained protein samples shouldbe loaded in the following manner:

Group A

Lane 1 Load 20µl of Tube A standard protein markersLane 2 Load 20µl of Tube B unknown protein 1Lane 3 Load 20µl of Tube C unknown protein 2Lane 4 Load 20µl of Tube D unknown protein 3

Group B

Lane 6 Load 20µl of Tube A standard protein markersLane 7 Load 20µl of Tube B unknown protein 1Lane 8 Load 20µl of Tube C unknown protein 2Lane 9 Load 20µl of Tube D unknown protein 3

RUNNING THE GEL

1. After the samples are loaded, carefully snap the cover all the waydown onto the electrode terminals. On EDVOTEK® electrophoresisunits, the black plug in the cover should be on the terminal with theblack dot.

2. Insert the plug of the black wire into the black input of the powersupply (negative input). Insert the plug of the red wire into the redinput of the power supply (positive input).

Volts Recommended Time

Minimum Optimal

125 40 min 1.0 hrs

70 60 min 1.5 hrs

Time and Voltage3. Set the power supply at the required voltage and run the

electrophoresis for the length of time as determined byyour instructor. When the current is flowing, you shouldsee bubbles forming on the electrodes. The sudsing isdue to the SDS in the buffer.

4. After the electrophoresis is finished, turn off power, unplugthe unit, disconnect the leads and remove the cover.


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Staining the Gel - OPTION #1

STAINING WITH PROTEIN INSTASTAIN® IN ONE EASY STEP(RECOMMENDED)

Protein polyacrylamide gels can be stained with Protein InstaStain®cards in one easy step.

1. After electrophoresis, carefully open the gel cassette and allow thegel to remain on one of the plates.

2. Submerge the gel and plate in a small tray with 100ml of fixativesolution. (Use enough solution to cover the gel.)

3. Lay the cassette down and remove the front plate by placing aspatula or finger at the top edge, near the sample wells, and lifting itaway from the larger back plate. In most cases, the gel will stay onthe back plate. If it partially pulls away with the front plate, let it fallonto the back plate.

4. Slide the gel into a staining tray.

If the gel sticks to the plate, pipet some of prepared staining solutiononto the gel and gently nudge the gel off the plate.

5. Gently float a sheet of Protein InstaStain® with the stain side (blue) inthe liquid. Cover the gel to prevent evaporation.

6. Gently agitate on a rocking platform for 1-3 hours or overnight.

7. After staining, Protein bands will appear medium to dark blueagainst a light background* and will be ready for excellent photo-graphic results. NO DESTAINING IS REQUIRED.

*If the gel is too dark, destain in several changes of fresh destainsolution until the appearance and contrast of the protein bandsagainst the background improves.

Storing the Gel

The gel should be stored in a mixture of 50ml of distilled water containing6ml of acetic acid and 3ml of glycerol overnight.

For permanent storage, the gel can be dried between two sheets ofcellophane (saran wrap) stretched in an embroidery hoop. Air dry thegel for several days until the gel is paper thin. Cut the "extra" saran wrapsurrounding the dried gel. Place the dried gel overnight between twoheavy books to avoid curling. Tape it into a laboratory book.

NOTE:

Polyacrylamide gels arevery thin and fragile.Use care in handling toavoid tearing the gel.

Proteins in this experimentare prestained and do notrequire staining. Proteinmolecular weights can bedetermined withoutstaining the gels.However, staining withenhance the proteinbands.

Fixative and DestainingSolution for each gel

(100ml)

50ml Methanol10ml Glacial Acetic Acid40ml Distilled Water




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Gentle agitation orincubation on a slowrotating or shakingplatform will acceleratethe destain time.

STAINING WITH PROTEIN PLUS™ STAIN (LIQUID)

1. Use a small plastic tray or large weigh boat for staining the gel indi-vidually.

2. Remove the gel cassette from the electrophoresis apparatus and blotoff excess buffer with a paper towel.

3. Lay the cassette down and remove the front plate by placing aspatula or finger at the top edge, near the sample wells, and lifting itaway from the larger back plate. In most cases, the gel will stay onthe back plate. If it partially pulls away with the front plate, let it fallonto the back plate.

4. Slide the gel into a staining tray.

If the gel sticks to the plate, pipet some of prepared staining solutiononto the gel and gently nudge the gel off the plate.

5. Cover the gel with 50ml of prepared staining solution. The gel shouldbe submerged in stain.

6. Cover the staining tray with plastic wrap to prevent evaporation.Stain for approximately two hours. The gel can be stained in a fewhours or overnight but will require a longer destain. (Note: The gelwill shrink.)

Destaining the gel

7. Pour off the stain from the gel in the staining tray.

8. Add 100ml of destaining solution. The gel should be submergedunder destaining solution.

9. Destain overnight (12 to 14 hours).

10. Remove the gel from the destaining liquid.

11. View the gel on a white light source. Photography of protein bands isoptional.

12. Examine the protein bands from the various samples and approxi-mate their molecular weights by comparison with the standardprotein markers.

Storing the Gel

The gel should be soaked in a mixture of 50ml of distilled water containing6ml of acetic acid and 3ml of glycerol overnight. The gel can be stored inthis solution or dried as described on page 14.

Staining the Gel - OPTION #2

Useful Hint!


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Determination of Molecular Weights

If measurements are taken directly from the gel, skip steps 1 and 2.

1. Take a transparent sheet, such as cellulose acetate (commonlyused with overhead projectors) and lay it over the wrapped gel.

2. With a felt-tip pen, carefully trace theoutlines of the sample wells. Thentrace over all the protein bands onthe gel.

3. Measure the migration distance, incentimeters (to the nearest millimeter)of every major band in the gel.(Ignore the faint bands, refer toIdealized Schematic.) All measure-ments should be from the bottom ofthe sample well to the bottom of theprotein band.

4. Using semi-log graph paper, plot themigration distance or Rf of eachstandard protein on the non-logarith-mic x-axis versus its molecular weighton the logarithmic y-axis. Chooseyour scales so that the data points arewell spread out. Assume the secondcycle on the y-axis represents 10,000to 100,000 (see example at left).

5. Draw the best average straight linethrough all the points. This line shouldhave an equal number of pointsscattered on each side of the line. Asan example, refer to the figure at left.This method is a linear approximation.

6. Using your standard graph, determinethe molecular weight of the threeunknown proteins. This can be doneby finding the Rf (or migration dis-tance) of the unknown band on the x-axis and drawing a straight verticaluntil the standard line is intersected. Astraight line is then made from theintersection across to the y-axis wherethe approximate molecular weightcan be determined.

In the above example, the standard molecular weights are:

94,00067,00043,00036,000

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Centimeters

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Bovine Serum Albumin

Ovalbumin

Lactate Dehydrogenase

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1. The migration rate of glutamate dehydrogenase is very similar to themigration rate of b-amylase during SDS polyacrylamide gel electro-phoresis. Yet the native molecular weight of glutamate dehydroge-nase is 330 kDa and that of β-amylase is 206 kDa. Explain.

2. Many genes have been cloned and sequenced. The precise aminoacid sequence of a polypeptide can be determined from the DNAsequence and the molecular weight can be calculated. Can areasonable estimate of the native molecular weight of a protein bedetermined from the sequence of their structural genes? Why?

3. IgG contains 2 small and 2 large polypeptide chains. A preparationof IgG was incubated with SDS, heated and submitted to SDS poly-acrylamide gel electrophoresis. One major band near the top of thegel was observed after staining. Explain.

4. Glutamate dehydrogenase can have a native molecular weight of2 x 106 in concentrated solutions. Upon the addition of NADH andglutamate, the native molecular weight is 330,000. Explain thesephenomena.

5. A purified, active preparation of carbonic anhydrase was submittedto native polyacrylamide gel electrophoresis at alkaline pH. Threemajor bands were observed after staining. The same preparation ofprotein was denatured and submitted to SDS-polyacrylamide gelelectrophoresis. One band was observed after staining. Explainthese results.

6. A glycoprotein consisting of a single polypeptide chain and over 40%(by weight) of N-acetylglucosamine, mannose and sialic acid wasfound to have a native molecular weight of 75,000 by several analyti-cal methods. However, analysis by SDS polyacrylamide gel electro-phoresis gave molecular weight of 100,000. Explain.

Study Questions


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19EDVO-Kit # 153 Determination of Protein Molecular Weight

Technical ServiceDepartment

FAX: (301) 340-0582web: www.edvotek.comemail: [email protected]

Please have the following information:

• The experiment number and title• Kit Lot number on box or tube• The literature version number (in lower right corner)• Approximate purchase date

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Notes to the Instructor

HOW THIS EXPERIMENT IS ORGANIZED

This experiment module contains biologicals and reagents for six (6) groupssharing three (3) polyacrylamide gels (2 groups per gel). Enough buffer isincluded for three (3) vertical electrophoresis units (Model MV-10 or equiva-lent). Additional electrophoresis buffer is required for more than three units.

Note: Polyacrylamide gels are not included. You may choose topurchase precast gels (Cat. #s 651 or 652).

The experimental procedures consist of two major parts:1) separation of proteins on polyacrylamide gels,2) determination of molecular weight of unknown proteins.

The staining of protein bands can be conducted using either ProteinInstaStain®, a new state-of-the-art method of staining, or by conventionalliquid staining with Protein Plus™ stain.

Protein InstaStain® is a proprietary staining method available exclusivelyfrom EDVOTEK®. You may wish to compare the staining methods by as-signing various student groups to use different staining methods.

APPROXIMATE TIME REQUIREMENTS FORPRE-LAB AND EXPERIMENTALPROCEDURES

1. Pre-lab preparations will require approxi-mately 20 minutes on the day of the lab.

2. Students will require approximately 15 min-utes to heat samples and load the gel. Prac-tice gel loading may require an additional15 minutes if performed the same day of thelab.

3. Electrophoresis will require approximately1 to 1.5 hours, depending on the voltage.


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PreLab Preparations

RECONSTITUTION OF PRESTAINED LYOPHILIZEDPROTEIN SAMPLES (LYPHOPROTEINS™)

Each tube contains enough material for loading 6 wells.

1. Add 130µl of distilled or deionized water to each tube (A-E) and allowthe samples to hydrate for several minutes. Vortex or mix vigorously.

2. Bring a beaker of water, covered with aluminum foil, to a boil. Re-move from heat.

3. Make sure the sample tubes A through E are tightly capped and welllabeled. The bottom of the tubes should be pushed through the foiland immersed in the boiling water for 10 minutes. The tubes should bekept suspended by the foil.

4. Samples can be aliquoted for each of the 6 student groups, orstudents can share the rehydrated sample stock tubes. Have studentsload samples onto the polyacrylamide gel while the samples are stillwarm to avoid aggregation. The volume of sample to load per well is20µl.

5. Store any unused portion of reconstituted sample at -20°C and repeatsteps 2 and 3 when using samples at a later time.

PREPARING ELECTROPHORESIS BUFFER

Prepare the electrophoresis buffer by adding and mixing 1 part Tris-Glycine-SDS 10x buffer concentrate to 9 parts distilled water.

The approximate volume of 1x electrophoresis buffer required for ED-VOTEK® Protein Vertical Electrophoresis units are listed in the table below.The buffer should just cover the back plate of the gel cassette.

Concentrated Distilled Total Buffer (10x) + Water = Volume

58ml 522ml 580ml

53ml 477ml 540ml

EDVOTEKModel #

MV-10

MV-20

Tris-Glycine-SDS Electrophoresis (Chamber) Buffer


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Pre-Lab Preparations

ELECTROPHORESIS TIME AND VOLTAGE

Your time requirements will dictate the voltage and the lengthof time for your samples to separate by electrophoresis.Approximate recommended times are listed in the table atleft.

PREPARING STAINING AND DESTAINING SOLUTIONS

The stock solution is used for staining with Protein InstaStain®and for preparation of Protein Plus™ Stain. Choose eithermethod for staining the gels.

1. Solution for staining with Protein InstaStain®

• Prepare a stock solution of Methanol and GlacialAcetic Acid by combining 180ml Methanol, 140mlDistilled water, and 40ml Glacial Acetic Acid.

• No destaining is required.

2. Protein Plus Staining Solution

• Combine and mix 270ml of Methanol/Glacial AceticAcid/Distilled water stock solution with the entirecontents of the Protein Plus™ stain.

3. Destaining Solution

• Use the stock solution of Methanol, Glacial Aceticacid and distilled water to destain the gels.

Volts Recommended Time

Minimum Optimal

125 30 min 1.0 hrs

70 40 min 1.5 hrs

Time and Voltage


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Idealized Schematic of Results

The idealized schematic below shows the relative positions of the denotedprotein bands, but are not drawn to scale. Faintly stained bands ob-tained from some samples are minor contaminants. Do not measurethese bands during analysis. Error can be ± 10% by this method.

DenaturedLanes Sample Protein Molecular

Weight*

1 & 6 A Standard Markers See figure2 & 7 B Unknown Protein 1 68,0003 & 8 C Unknown Protein 2 58,0004 & 9 D Unknown Protein 3 50,000


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Study Questions and Answers

1. The migration rate of glutamate dehydrogenase is very similar to themigration rate of b-amylase during SDS polyacrylamide gel electro-phoresis. Yet the native molecular weight of glutamate dehydroge-nase is 330 kDa and that of βββββ-amylase is 206 kDa. Explain.

Glutamate dehydrogenase is a hexamer with a molecular weight of300,300 (+/- 2000), β-amylase is a tetramer with a molecular weight of206,000. Upon denaturation with SDS and 2-mercaptoethanol, 6subunits of approx. 50,000 will be generated from glutamate dehydro-genase. Likewise, the native β-amylase, which has 4 subunits, will yielda protein band of approx. 50,000

2. Many genes have been cloned and sequenced. The precise aminoacid sequence of a polypeptide can be determined from the DNAsequence and the molecular weight can be calculated. Can areasonable estimate of the native molecular weight of a protein bedetermined from the sequence of their structural genes? Why?

The native molecular weight of a subunit can be determined by thismethod, however often proteins are aggregates of polypeptidechains. Approximately 1% of protein mass consists of carbohydrateand the structure and mass of the oligosaccharide cannot be antici-pated by DNA sequencing. Also, DNA does not yield information onenzyme cofactors.

3. IgG contains 2 small and 2 large polypeptide chains. A preparationof IgG was incubated with SDS, heated and submitted to SDS poly-acrylamide gel electrophoresis. One major band near the top of thegel was observed after staining. Explain.

Disulfide links are still intact. SDS does not cause the cleavage ofcovalent bonds.

4. Glutamate dehydrogenase can have a native molecular weight of2 x 106 in concentrated solutions. Upon the addition of NADH andglutamate, the native molecular weight is 330,000. Explain thesephenomena.

Concentrated solutions of the protein tend to polymerize, formingchains of approximately 6 enzyme molecules. The substrates NADHand glutamate are bound, causing conformational changes in theprotein and disaggregation to the 330,000 molecular weight enzyme.

5. A purified, active preparation of carbonic anhydrase was submittedto native polyacrylamide gel electrophoresis at alkaline pH. Threemajor bands were observed after staining. The same preparation ofprotein was denatured and submitted to SDS-polyacrylamide gelelectrophoresis. One band was observed after staining. Explain theseresults.

Carbonic anhydrase has 3 major isoenzymic forms, differing in aminoacid sequence and net charge but all having similar molecularweights.


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Study Questions and Answers

6. A glycoprotein consisting of a single polypeptide chain and over 40%(by weight) of N-acetylglucosamine, mannose and sialic acid wasfound to have a native molecular weight of 75,000 by several analyti-cal methods. However, analysis by SDS polyacrylamide gel electro-phoresis gave molecular weight of 100,000. Explain.

SDS-polyacrylamide gel electrophoresis gives anomalous molecularweight values for proteins having high percentages of carbohydrate.These proteins tend to bind less SDS than normal, which tends todecrease the charge to mass ratio.

Material Safety Data SheetMay be used to comply with OSHA's Hazard Communication

Standard. 29 CFR 1910.1200 Standard must be consulted forspecific requirements.

IDENTITY (As Used on Label and List) Note: Blank spaces are not permitted. If any item is not applicable, or no information is available, the space must be marked to indicate that.

Section IManufacturer's Name

Section II - Hazardous Ingredients/Identify Information

Emergency Telephone Number

Telephone Number for information

Date Prepared

Signature of Preparer (optional)

Address (Number, Street, City, State, Zip Code)

EDVOTEK, Inc.

14676 Rothgeb DriveRockville, MD 20850

Hazardous Components [Specific Chemical Identity; Common Name(s)] OSHA PEL ACGIH TLV

Other Limits Recommended % (Optional)

(301) 251-5990

(301) 251-5990

®

Boiling Point

Section III - Physical/Chemical Characteristics

Unusual Fire and Explosion Hazards

Special Fire Fighting Procedures

Vapor Pressure (mm Hg.)

Vapor Density (AIR = 1)

Solubility in Water

Appearance and Odor

Section IV - Physical/Chemical CharacteristicsFlash Point (Method Used)

Extinguishing Media

Flammable Limits UELLEL

Melting Point

Evaporation Rate(Butyl Acetate = 1)

Specific Gravity (H 0 = 1) 2

10x Tris Glycine Buffer

09-15-2002

Glycine (C2H5NO2) ---------------------No data-----------------CAS # 56-40-6TrisCAS # 77-86-1

No data

No data

No data

No data

No data

No data

Soluble

Clear, no odor

No data No data No data

Water spray, carbon dioxide, dry chemical powder or appropriate foam

Wear SCBA and protective clothing

May emit toxic fumes

Stability

Section V - Reactivity DataUnstable

Section VI - Health Hazard Data

Incompatibility

Conditions to Avoid

Route(s) of Entry: Inhalation? Ingestion?Skin?

Other

Stable

Hazardous Polymerization

May Occur Conditions to Avoid

Will Not Occur

Health Hazards (Acute and Chronic)

Carcinogenicity: NTP? OSHA Regulation?IARC Monographs?

Signs and Symptoms of Exposure

Medical Conditions Generally Aggravated by Exposure

Emergency First Aid Procedures

Section VII - Precautions for Safe Handling and UseSteps to be Taken in case Material is Released for Spilled

Waste Disposal Method

Precautions to be Taken in Handling and Storing

Other Precautions

Section VIII - Control Measures

Ventilation Local Exhaust Special

Mechanical (General)

Respiratory Protection (Specify Type)

Protective Gloves

Other Protective Clothing or Equipment

Work/Hygienic Practices

Eye Protection

Hazardous Decomposition or Byproducts

X

Strong oxidizing agents

Carbon monoxide, carbon dioxide, nitrogen oxides

X

Yes Yes Yes

May cause irritation to eyes, skin, and mucous membranes.

No data No data No data No data

Irritation

Unknown

Skin/eye contact: flush w/ water. Inhalation: remove to fresh air.Ingestion: Seek medical attention

Wear respirator and protective clothing. Mop up with absorbant material and dispose of properly.

Burn in chemical incinerator equipped w/ afterburner and scrubber. Follow all state, federal, and local regulations.

Avoid contact, keep away from heat.

None

NoYes

NoneNone

Rubber or PE Safety goggles

Lab coat, coveralls

Prevent contact








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Practice Gel Loading Solution

09-19-2002

This product contains no hazardous materials as defined by the OSHA Hazard CommunicationStandard.

No data

No data

No data

No data

No data

No data

Soluble

Blue liquid, no odor

No dataNo data No data

Dry chemical, carbon dioxide, water spray or foam

Use agents suitable for type of surrounding fire. Keep upwind, avoid

breathing hazardous sulfur oxides and bromides. Wear SCBA.

Unknown

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None

None

Sulfur oxides, and bromides

X None

Yes Yes Yes

Acute eye contact: May cause irritation. No data available for other routes.

No data available

May cause skin or eye irritation

None reported

Treat symptomatically and supportively. Rinse contacted area with copious amounts of water.

Wear eye and skin protection and mop spill area. Rinse with water.

Observe all federal, state, and local regulations.

Avoid eye and skin contact.

None

Yes None

Yes None

Yes Splash proof goggles

None required

Avoid eye and skin contact








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Protein InstaStain

09-19-2002

Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100%CH3OH

65°C

96mmHg

1.11

.79

N/A

4.6

Complete (100%)

chemical bound to paper, no odor

(closed cup) 12°C 6.0% 36%

Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)

Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea

Vapors may flow along surfaces to distant ignition sources.

Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None


Carbon monoxide, Carbon dioxide, Sulfur oxides

X None

Rubber boots

Avoid prolonged or repeated exposure

Yes Yes YesIrritating to eyes, skin, mucous membranes and upper respiratory tract.

Chronic exposure may cause lung damage or pulmonary sensitization


Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache

No data

Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amounts of water or milk. Do not induce vomiting.

Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burn in chemical incinerato equipped w/ an afterburner and scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Strong sensitizer

NIOSH/MSHA approved respirator

No Chem fume hood

No None

Rubber Splash-proof goggles








Date Prepared



EDVOTEK, Inc.




(301) 251-5990

(301) 251-5990

®

Boiling Point






Solubility in Water

Appearance and Odor


Extinguishing Media


Melting Point



Protein Plus Stain

09-19-2002

Methanol (Methyl Alcohol) 200ppm 200ppm No data 90%-100%CH3OH

65°C

96mmHg

1.11

.79

N/A

4.6

Complete (100%)

Blue liquid/alcoholic, pungent odor

(closed cup) 12°C 6.0% 36%

Use alcohol foam, dry chemical or carbon dioxide. (Water may be ineffective)

Wear SCBA with full facepiece operated in positive pressure mode. Move containers from firearea

Vapors may flow along surfaces to distant ignition sources.

Close containers exposed to heat may explode. Contact w/ strong oxidizers may cause fire.

Stability



Incompatibility

Conditions to Avoid


Other

Stable



Will Not Occur









Other Precautions





Protective Gloves



Eye Protection


X None


Carbon monoxide, Carbon dioxide, Sulfur oxides

X None

Rubber boots

Avoid prolonged or repeated exposure

Yes Yes YesIrritating to eyes, skin, mucous membranes and upper respiratory tract.

Chronic exposure may cause lung damage or pulmonary sensitization


Respiratory tract: burning sensation. Coughing, wheezing, laryngitis, shortness of breath, headache

No data

Flush skin/eyes w/ large amounts of water. If inhaled, remove to fresh air. Ingestion: give large amounts of water or milk. Do not induce vomiting.

Evacuate area. Wear SCBA, rubber boots and rubber gloves. Mop up w/ absorptive material and burn in chemical incinerato equipped w/ an afterburner and scrubber.

Observe all federal, state, and local laws.

Wear protective gear. Avoid contact/inhalation.

Strong sensitizer

NIOSH/MSHA approved respirator

No Chem fume hood

No None

Rubber Splash-proof goggles

edvo-kit - 154.68.126.6

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