Fralin Life Science Institute Protein Electrophoresis Kit
Fralin Life Science InstituteProtein Electrophoresis KitINFORMATION MANUALKristi DeCourcy & Erin DolanFralin Life Science InstituteVirginia TechAugust 2008
TABLE OF CONTENTSpageIntroductionCalendar of activities . .Introduction to the Protein Electrophoresis Kit . .Protein electrophoresis kit contents . . .Important notes (Please read!) . . .Introduction to protein electrophoresis . . .ExperimentOverview of the experimental procedure . .Choosing samples .Safety note .Preparing samples for SDS-PAGE .Running a SDS-PAGE gel .Staining, destaining, and drying the gel .Gel analysis . Clean up . .AppendicesAppendix table of contents .Pipettor directions . .Setting up a SDS-PAGE gel for electrophoresis .Power supply directions . . .Molecular weight standards . . .Practice gel analysis . . . .Conceptual review materials Technical review materials .Real world applications (scenarios) . .Protein electrophoresis kits available commercially Some information on muscle proteins Some information on evolutionary relationships . Virginia Standards of Learning & National Science Education StandardsSources for materials and equipment included in this kit Solutions used in the kit . . . .References . . . .Directions for return shipping .Semilog graph paper 62636465ACKNOWLEGMENTSWe would like to thank the teachers who have made many helpful suggestions for improvingthe kit and the manual, including Kristin Jenkins, Jill Peters, John McLaughlin, StevenScheidell, Jim Meadows, Greg Deskins, and Adrienne Warren. We would also like toacknowledge the Biotechnology Explorer Program at Bio-Rad, with whom we traded manyideas during the development of this Biotech-in-a-Box kit and their classroom kit.2
Introduction: CalendarCalendar of Activities: Electrophoresis of Protein SamplesClassschedule45-50 minuteperiods withno blocks45-50 minuteperiods plusone block (90120 minutes)All blocks (90120 minutes)Day 1Day 2Day 3Only part of period isneeded Introduce lab (15 min) Prepare protein samples(15 min) Teacher or trusted students set upelectrophoresis chambers before or earlyin class Load and run (45 min) Stain (20 min) Destain (at least 20min, but leave indestain overnight) Begin analysisBlock day Teacher or trustedstudents set upelectrophoresis chambersbefore or early in class Introduce lab (15 min) Prepare protein samples(15 min) Load and run (45 min)Entire block Teacher or trustedstudents set upelectrophoresis chambersbefore or early in class Introduce lab (15 min) Prepare protein samples(15 min) Load and run (45 min)Regular day Stain (20 min) Destain (at least 20 min, but leave indestain overnight) Begin analysisRegular day Complete analysis Wrap-up (Optional) Begindrying gels. They willbe dry within 24-48hours for long-termstorage.Day 4 Complete analysis Wrap-up (Optional) Begin dryinggels. They will be drywithin 24-48 hours forlong-term storage.Entire block Stain (20 min) Destain (at least 20 min, but leave indestain throughout period) Analysis Wrap-up (Optional) Begin drying gels. They will bedry within 24-48 hours for long-termstorage. Bands may be more visible if gelsare destained overnight prior to drying.Optional stopping points:1. After preparing protein samples but before loading gel. Protein samples will keep overnight in the refrigerator. Allow samples to warm toroom temperature before loading gel.2. After running and staining gel while destaining. Store gel just barely covered with destain in dish (e.g., weigh boat, plastic container, etc.)covered with plastic wrap. The gel will keep overnight. No need to refrigerate. Do not leave gel soaking in stain overnight – it willshrink!3. After destaining gel. Store gel in water in a plastic wrap-covered weigh boat or plastic container with lid. The gel will keep for several daysprior to drying. No need to refrigerate.3
IntroductionINTRODUCTION TO THE PROTEIN ELECTROPHORESIS KITThe Protein Electrophoresis Kit from the Fralin Biotechnology Center contains all thematerials needed to prepare samples, run SDS-PAGE gels, visualize the proteins on thegels, and dry the gels to preserve them. The borrower must provide the sample materials(fish, seafood, meat, etc.). The kit is available from the Center for a 2-week loan period.Electrophoresis of proteins using SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gelelectrophoresis) is similar in principle to electrophoresis of DNA in agarose, but there aresome significant differences. One of the primary differences is that acrylamide in its liquidform is a neurotoxin, and, as such, is not suitable for classroom use. The gels will beprovided ready to use. Another difference is that the apparatus is slightly more complicatedto set up and run. Because of this, we ask that the instructor, or perhaps the instructor with afew trusted students, set up the gel boxes for use. The class can prepare the samples andload them on the gels, and then process the gels after the run.This manual is divided into three sections. The first section includes a calendar of activity, alist of kit contents, and a section on the theory of protein electrophoresis. The instructor candecide the complexity of the material to be presented to the students. The most importantpoints are highlighted and there are several overheads on the material. (The overheads arethe same as the figures in the text and will be sent in the kit.)The second section is the experimental section, containing an overview of the experiment,notes on the reagents used, and the student experimental procedure. The experimentalprocedure is broken into several sections; each section is preceded by information for theinstructor, including advanced preparation needed.The final section is the Appendix, which contains everything else, including gel boxassembly, scenarios, pipettor directions, conceptual and technical review material, practicegel analysis, sources for the supplies and equipment, references, and information on muscleproteins and on evolutionary relationships.If you come up with any other scenarios or study questions, please send us a copy and we’llincorporate them in the manual as well.Feel free to call Drs. Kristi DeCourcy or Erin Dolan at the Center with any questions aboutthe material. Corrections and suggestions will be gratefully received. Kristi’s phone is (540)231-7959 (email is decourcy@vt.edu) and Erin’s is (540) 231-2692 (email edolan@vt.edu).New sponsor!!The Fralin Outreach Program, specifically Biotech-in-a-Box, has received a grant from theVirginia Council on Advanced Technology Skills (VCATS), in partnership with the VirginiaBiotechnology Association (VaBIO) and the Virginia Manufacturers Association (VMA). Weare very grateful for their support, which will enable up to purchase new equipment and toexpand the program.Manuals on CD!Instead of hard copies, this manual (and the other three as well) has been sent to you on aCD. Since there are few changes in the manual from year-to-year, this seemed like the righttime to make a change. This should make it easier to print materials from the manual. If youwould infinitely prefer a hard copy, please contact me with a request.4
Introduction: Kit ContentsPROTEIN ELECTROPHORESIS KIT CONTENTSItempower supplygel boxesbuffer damsgel loading guidesFisher micropipettor (5-40 µl)10-µl fixed volume pipettor20-µl fixed volume pipettoryellow tips for micropipettorstube racksfloating tube racksscissorsforcepsspatulasrazor bladesplastic boxesQuantity14441441 bag102101051 pk5weigh boatsmicrocentrifuge tubescolored microcentrifuge tubesmarker penslabel tapeboiling caps1-ml graduated transfer pipettestransfer pipettesgel drier rackgel drier framesgel drier clampsKimwipes glovesextra gel cassette (for demo)50-ml graduated cylinderOverheads (envelope)101 pack1 pack22 50202015201 box1 box111The quantities of the following items vary with the number of gels the instructor plans to runwith his or her classes. The maximum number of gels available with the kit from the Center isfive, although additional gels may be purchased by the instructor from Bio-Rad, if desired(see page 61 for ordering information). Fralin will provide, upon request, buffers andmaterials for up to 10 additional gels.The sample buffer provided is enough to prepare 50 samples. Additional sample buffer willbe available if you plan to prepare more samples. Please make this decision before the kit isshipped, so we can avoid additional shipping charges. Ten samples may be run on each gel(actually, 9 plus a molecular weight standard).Itempre-cast gelsLaemmli sample buffermolecular weight markersgel running bufferCoomassie stain solutiondestain solutiongel drying solutioncellophane sheetsQuantity525 ml20 µl/gel400 ml/gel50 ml/gel100 ml/gel40 ml/gel2/gel5storage conditionsrefrigeratorroom temperaturefreezerroom temperatureroom temperatureroom temperatureroom temperatureroom temperature
Introduction: Important NotesIMPORTANT NOTES (Please read!) Please remember that this kit is shipped with 5 gels and the materials to run them(buffers, cellophane sheets, etc.). If you wish to run more gels while you have the kit, youmust purchase them from Bio-Rad directly, and let the Center know ahead of time thenumber of extra gels for which you will need materials (maximum: materials for 10additional gels). If you decide to purchase more gels, please remember that there are 4gel boxes in the kit, so the most you can run at a time is 4 gels (or 8, if you run 2 gels perbox). On the checklist, if something is not shaded in the “repacked” column, that means that itis not an expendable item, and all should be returned, barring a problem, (e.g., if yourdog eats a tube rack). On the checklist, if something is shaded in the “repacked” column, that means that it is anexpendable item. It is not expected that you will return all of these items, but please returnany that are unused. Please do not mix used items with unused items! Please help uskeep our expendable costs down by returning unused items. Please rinse the gel boxes and accessories and allow them to air dry (if possible) beforerepacking them. A number of boxes have been returned with dried buffer residue all overthem. Please see clean-up directions later in manual. If there is a problem with a piece of equipment, please indicate which piece by puttinglabel tape on it. Please do not just say, “A gel box didn’t work,” or we have to spend timetesting every box. Please (!) keep to the schedule for return shipping! If you are late returning the kit, thenext teacher is the one who will suffer, and it’s not fair to any of us! Seal the trunks with the cable ties provided. Please make sure that the cable ties aresecure. Note that there is a right way and a wrong way to insert the tab in the cable tie.Look at the end you put the tab through. The tab should be put in from the side that issmooth with the tie, not the end that sticks out. If it is done the wrong way, the cable tiewill open when you pull on it. Please test the cable tie by pulling to be sure that you’vedone it correctly. Be certain that the shipping label is placed on the trunk securely in the plastic sleeve. Please be careful with repacking. Use the bubble bags, and place heavy items on thebottom of the trunk. Please make sure that the lids are on the gel boxes for returnshipping or the leads can be broken off. Please, do not use Styrofoam peanuts!!6
Introduction: Protein ElectrophoresisINTRODUCTION TO PROTEIN ELECTROPHORESISGeneral principles of electrophoresisElectrophoresis is the migration of charged molecules in an electric field toward theelectrode with the opposite charge (Figure 1). This technique is widely used to examineproteins and nucleic acids, answering a variety of questions. For example: How pure is this protein? How much DNA do I have? How big is this protein? Has my experiment resulted in any changes in my protein? What is the nucleotide sequence of this DNA?Although charged molecules would migrate to the opposing electrode if the electrophoresiswere performed in solution alone, electrophoresis is commonly performed bycathode (negative electrode)placing the sample in a matrix of eitheracrylamide or agarose. In gel electrophoresis, molecules separate based on theircharge and their mass. The gel matrix actsas a molecular sieve through which smallermolecules can move more quickly thanlarger molecules. The amount of sievingthat takes place during electrophoresis canbe increased or decreased by changingdirection ofthe concentration of agarose or acrylaprotein flowmide.--------------Acrylamide or agarose? anode (positive electrode)Figure 1. In electrophoresis, negatively chargedproteins or nucleic acids move toward the positivelycharged electrode.In general, DNA molecules are orders ofmagnitude larger than proteins. Thinkabout this: DNA codes for proteins. Codingfor each amino acid of a protein requires 3basepairs of DNA, plus all theDNAforthetranscriptioncontrols: signals for the gene toturn on and turn off, signals thatdetermine where transcription willstart and stop, etc.?Proteins range in size from severalkilodaltons (kDa) to hundreds or thousandsof kilodaltons. The nucleic acids we study are normally larger than a kilobase (kb), and eachkilobase is approximately 660 kilodaltons. For example, when cloning DNA, 2 kilobasepieces of DNA are frequently inserted in plasmids of 3 kilobases, giving a total plasmid lengthof 5 kilobases ( 3,300 kilodaltons or 3.3 million daltons).When the molecules to be separated are large (greater than 200 kDa), then agarose is thematrix of choice. This means that agarose gel electrophoresis is generally used forseparating nucleic acids and some larger proteins. Acrylamide is used for most proteinelectrophoresis and for separation of small DNA molecules (e.g., for DNA sequencing).7
Introduction: Protein ElectrophoresisDetermining the size of proteins using electrophoresisOne of the uses of gel electrophoresis is determining the molecular weight of a protein.(Determining the purity of a protein is a second major use.) Proteins will migrate in anacrylamide gel at a rate based both on their electric charge and on their mass. The ratio ofcharge to mass is called charge density. In order to use this technique for molecular weightdeterminations, the charge of the protein must be removed as a factor in the migration.This is accomplished by placing an ionic detergent in both the gel running buffer and thesample buffer. The detergent commonly used is sodium dodecyl sulfate (SDS). Most proteinsbind SDS in such a way that they migrate in a gel as if they have nearly identical chargedensities (all negative), so mass becomes the only factor in determining the migration rate ofthe protein. This technique is called SDS-PAGE (SDS-polyacrylamide gel electrophoresis).Although it will not be discussed here, PAGE is frequently performed without SDS. "Native"PAGE (native, because the proteins are in their native conformations) is used, for example,when it is important that the protein of interest not lose its structure or activity. Also SDS isnot used when sequencing nucleic acids, because, unlike proteins, most nucleic acids havethe same charge densities without adding SDS.SDS-PAGE using a discontinuous buffer systemOne final consideration is how one gets all the protein to enter the gel at the same time.Samples usually have microliter (µl) volumes, and not all the protein molecules will enter thegel at the same time.THE SHORT ANSWER: A "stacking" gel is poured on top of the "separating" gel. The stackinggel has a lower concentration of acrylamide and a different buffer molarity and pH than theseparating gel. As a result of the stacking gel, all the proteins in the sample enter theseparating gel simultaneously. Most, but not all, of the pre-poured gels offered by vendorshave stacking gels.RUNNING BUFFERTris-glycine, pH 8.3CATHODE-STACKING GEL125 mM Tris, pH 6.8SEPARATING GEL250 mM Tris, pH 8.8 ANODEFigure 2. A discontinuous gel system is one in which astacking gel is poured on top of the separating gel. Thestacking gel has a lower acrylamide concentration than theseparating gel and a lower pH than the buffer or separating gel.8THE LONG ANSWER: Thesolution is using a discontinuous buffer system which hasa "stacking" gel (Figure 2). Agel of lower acrylamide concentration, typically 3-4%, ispoured on top of the "separating" gel, typically 6-15%. Thestacking gel (pH 6.8) and theseparating gel (pH 8.8) aremade with Tris-HCl buffers.The running buffer is a Trisglycine buffer, pH 8.3. In anelectric field at pH 6.8, chlorideions migrate the most rapidly,proteins have intermediatemobility, and glycine migratesthe most slowly. As electrophoresis begins, the chlorideions move ahead, causing azone to form behind them in
Introduction: Protein Electrophoresiswhich there is reduced conductivity. Because of this reduced conductivity, the migration ofglycine is accelerated, so that it keeps up with the chloride. Basically, the proteins are"captured" in the zone between the chloride and the glycine in the order of their mobility, sothat the protein with the least mobility (the largest) will be closest to the glycine. In otherwords, the proteins are "stacked" in a very narrow band when they reach the separating gel.When this stack reaches the interface of the stacking and separating gels, the pH increasesto 8.8. At pH 8.8, the mobility of glycine increases and it passes the proteins and migratesdirectly behind the chloride ions. The higher percentage acrylamide in the separating gelresults in increased sieving of the proteins, which are no longer stacked, and the proteinsseparate according to their mass.A little historyFor a historical note, the gel system that we are using is credited to U.K. Laemmli, whose1970 Nature paper has probably the highest number of citations of any scientific paper.Laemmli used the buffer system developed by Ornstein and Davis. This overall system, aSDS-containing discontinuous gel with the Ornstein-Davis buffer system, is the predominantone used in slab gel electrophoresis.Preparing a polyacrylamide gelPolyacrylamide gels are formed by the polymerization of acrylamide monomers, and thecrosslinking of these polymers by bis-acrylamide (N,N'-methylene bisacrylamide). Theamount of sieving by the gel is controlled by adjusting the acrylamide concentration, withhigher concentrations of acrylamide resulting in increased sieving. For example, to separateproteins of 100 to 200 kDa, the acrylamide concentration should be 5%, but to separateproteins in the range of 20 to 40 kDa, the acrylamide concentration should be 15%. In theexperiment in this kit, you will be using a separating gel of 15% acrylamide, with a 4%
Store gel just barely covered with destain in dish (e.g., weigh boat, plastic container, etc.) covered with plastic wrap. The gel will keep overnight. No need to refrigerate. Do not leave gel soaking in stain overnight – it will shrink! 3. After destaining gel. Store gel in water in a plastic wrap-covered weigh boat or plastic container with lid.
protein:ligand, K eq [protein:ligand] [protein][ligand] (1) can be restated as, K eq 1 [ligand] p 1 p 0 (2) where p 0 is the fraction of free protein and p 1, the fraction of protein binding the ligand. Assuming low protein concentration, one can imagine an isolated protein in a solution of Nindistinguishable ligands. Under these premises .
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of protein assay for research applications. Protein assays based on these methods are divided into two categories: dye binding protein assays and protein a ssays based on alkaline copper. The dye binding protein assay s are based on the binding of protein molecules to Coomassie dye under acidic conditions.
protein, the USDA Choose My Plate food guidance system has identified nuts & seeds and beans & peas as plant-based protein foods. The following chart tells you how much of a plant-based protein food provides as much protein as an ounce of cooked meat, poultry or seafood. Ounce Equivalents for Protein Foods Meats 1 ounce cooked beef, pork or lamb
protein-protein interaction data and the protein sequences. In particular, our method as-sumes no knowledge of the 3D protein structure, or of the sites at which binding occurs. Our approach is based on the assumption that interaction sites can be described using a limited repertoire of conserved sequence motifs [11].
biological significance of protein complexation with RNA has been well recognized, the specific mecha-nism of protein–RNA interaction is not fully understood [10]. Measurement of sequence–specific DNA– protein and RNA–protein interactions is a key experimental procedure in molecular biology of gene regulation.
x Determine protein concentrations using the Biuret Protein Assay. BACKGROUND The determination of protein concentration is an essential technique in all aspects of protein studies and proteomics. This lab activity is designed to teach students the principl es behind a common protein es
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