Protein Gel Electrophoresis Technical Handbook
Western blottingProtein gel electrophoresistechnical handbook separate transfer detect
2Select precast gelComprehensive solutionsdesigned to drive your successPrepare samplesand select buffersSelect the standardChoose the electrophoresischamber system and power supplyRun the gelStain the gelPost stainContentsElectrophoresis overview4Select precast gelProtein gel electrophoresis is a simple way toseparate proteins prior to downstream detectionor analysis, and is a critical step in mostworkflows that isolate, identify, and characterizeproteins. We offer a complete array of productsto support rapid, reliable protein electrophoresisfor a variety of applications, whether it is thefirst or last step in your workflow. Our portfolioof high-quality protein electrophoresis productsunites gels, stains, molecular weight markers,running buffers, and blotting products foryour experiments.810Gel selection guideGelsPrepare samples and select buffers262829Sample prep kitsBuffers and reagentsBuffers and reagents selection guideSelect the standard3436Protein laddersProtein standards selection guideChoose the electrophoresis chamber system and power supply505158Electrophoresis chamber systemsElectrophoresis chamber system selection guidePower suppliesRun the gel5960Gel run conditionsTroubleshooting tipsStain the gel6263, 67, 69, 7071Protein stainsProtein stains selection guidesElectrophoretic staining technologyPost stain74Transfer and detectionAppendixand more, visit thermofisher.com/separateFor ordering information refer to page XX. For quick reference on the protocol please refer to page XX.7681Protocol quick referenceOrdering information For a complete listing of all available productsPrecast protein gelsSample preparation andelectrophoresis buffersProtein standardsElectrophoresis chambersystems and power suppliesElectrophoresisrun conditionsProtein gel stains3
4Select precast gelPrepare samplesand select buffersSelect the standardChoose the electrophoresischamber system and power supplyRun the gelStain the gelPost stainElectrophoresisSupport matrixElectrophoresis is defined as theTwo types of support matrices are commonly used intransport of charged moleculeselectrophoresis—polyacrylamide and agarose. The supportmatrices act as porous media and behave like a molecular sieve.through a solvent by an electricSeparation of molecules is dependent upon the gel pore size ofthe support matrix used. Agarose has a large pore size and is idealfield. Electrophoresis is a simple,for separating macromolecules such as nucleic acids and proteincomplexes. Polyacrylamide has a smaller pore size and is ideal forrapid, and sensitive analyticalseparating most proteins and smaller nucleic acids.tool for separating proteins andPolyacrylamide gel electrophoresisnucleic acids. Any charged ion or(PAGE)molecule will migrate when placed Polyacrylamide gels are generated by the polymerization ofacrylamide monomers. These monomers are crosslinked intoin an electric field. Most biologicallong chains by the addition of bifunctional compounds such as(bis), which react with the freemolecules carry a net charge at any N,N,-methylenebisacrylamidefunctional groups of the chain termini. The concentration ofacrylamide and bisacrylamide determines the pore size of the gel.pH other than at their isoelectricThe higher the acrylamide concentration, the smaller the pore size,resulting in resolution of lower molecular weight molecules andpoint and will migrate at a ratevice versa.proportional to their charge density. PAGE allows one to separate proteins for different applicationsThe mobility of a biological moleculethrough an electric field will dependon the following factors:based on: The acrylamide matrix Buffer systemsElectrophoresis conditionsLinear vs. gradient gelsThe separation of molecules is dependent on the electrophoresisconditions. Electrophoresis can be performed under thefollowing conditions:Gels that have a single acrylamide percentage are referred to aslinear gels, and those with a range are referred to as gradientgels. The advantage of using a gradient gel is that it allows theseparation of a broader range of proteins than a linear gel.Continuous vs. discontinuous gelsResearchers occasionally refer to gels as continuous ordiscontinuous. A continuous gel is a gel that has been formedfrom a single acrylamide solution in the entire gel cassette.A discontinuous gel is formed from two acrylamide solutions, asmall, low-percentage stacking gel where the protein wells reside,and a larger portion of gel that separates the proteins. In thetraditional Tris-glycine protein gel system, the proteins are stackedin the stacking gel between the highly mobile leading chloride ions(in the gel buffer) and the slower, trailing glycine ions (in the runningbuffer). The reason for using the stacking gel is to improve theresolution of the bands in the gel. These stacked protein bandsundergo sieving once they reach the separating gel.Mini vs. midi protein gelsCommercial gels are available in two size formats, minigels andmidigels. Both gels have similar run lengths, but midigels are widerthan minigels, allowing midigels to have more wells or larger wells.The additional wells in the midigels permit more samples or largesample volumes to be loaded onto one gel.Denaturing conditionsElectrophoresis is performed under denaturing conditions usingan anionic detergent such as sodium dodecylsulfate (SDS).SDS denatures and unfolds the protein by wrapping around thehydrophobic portions. SDS binds at a ratio of 1.4 g SDS pergram of protein. The resultant SDS-protein complexes are highlynegatively charged and are resolved in the gel based on their size.Nondenaturing (native) conditionsElectrophoresis is performed under nondenaturing (native)conditions using buffer systems that maintain the native proteinconformation, subunit interaction, and biological activity. Duringnative electrophoresis, proteins are separated based on theircharge to mass ratios.Reducing conditionsElectrophoresis is performed under reducing conditions usingreducing agents such as dithiothreitol (DTT), β-mercaptoethanol(β-ME) or tris(2-carboxyethyl)phosphine (TCEP).The reducing agents completely unfold the denatured proteinsinto their subunits by cleaving the disulfide bonds betweencysteine residues.Buffer systems Electrophoresis conditionsElectrophoresis is performed using continuous or discontinuousbuffer systems. A continuous buffer system utilizes only onebuffer in the gel and running buffer. A discontinuous buffer systemutilizes a different gel buffer and running buffer1. This system mayalso use two gel layers of different pore sizes and different buffercomposition (the stacking and separating gel). Electrophoresisusing a discontinuous buffer system results in concentration of thesample and higher resolution. Field strength Net charge on the molecule Size and shape of the molecule Ionic strengthDid you know?Arne Tiselius won theNobel Prize in Chemistryfor electrophoretic analysisof serum proteins in 1948.Reference Properties of the matrix through whichthe molecules migrate (e.g., viscosity,pore size)The acrylamide matrix1. Ornstein L (1964) Disc electrophoresis. 1. Background and theory. Ann N Y Acad Sci 121:321-349.Mini Gel TankPrecast protein gelsSample preparation andelectrophoresis buffersProtein standardsElectrophoresis chambersystems and power suppliesElectrophoresisrun conditionsProtein gel stains5
6Select precast gelComparison ofdiscontinuous buffersystemsSDS-PAGE utilizes a discontinuous buffer system to concentrateor “stack” samples into a very sharp zone in the stacking gel at thebeginning of the run. In a discontinuous buffer system, the primaryanion in the gel is different (or discontinuous) from the primaryanion in the running buffer. Both the Invitrogen NuPAGE systems(Bis-Tris and Tris-acetate gels) and the Laemmli (Tris-glycine)system are examples of discontinuous buffer systems and workin a similar fashion. However, the NuPAGE system operates at alower pH as a result of the proprietary ions that are in the system.In a Tris-glycine system (Figure 1), three ions are primarily involved: C hloride (–), supplied by the gel buffer, serves as the leadingion because it has the highest attraction to the anode relative toother anions in the system. Glycine (–), the primary anion provided by the running buffer,serves as the trailing ion, because it is only partially negativelycharged and remains behind the more highly charged chlorideions in a charged environment. Tris base ( ), is a common ion present in both the gel and therunning buffers. During electrophoresis, the gel and buffer ionsin the Tris-glycine system form an operating pH of 9.5 in theseparating region of the gel.In the case of the Bis-Tris system (Figure 2), three ions areprimarily involved: Chloride (–) supplied by the gel buffer, serves as the fast-movingleading ion. MES or MOPS (–) (depending on the running buffer choice)serves as the trailing ion. MES: 2-(N-morpholino) ethane sulfonic acid MOPS: 3-(N-morpholino) propane sulfonic acid Bis-Tris ( ) acts as the common ion present in the gel whileTris ( ) is provided by the running buffer.With the Tris-acetate system (Figure 3), three ions areprimarily involved: Acetate (–), the leading ion from the gel buffer Tricine (–), the trailing ion from the running buffer Tris ( ), the common ion (in both gel and running buffer)This system also operates at a significantly lower pH than the Trisglycine system, resulting in less gel-induced protein modifications.The diagrams below (Figures 1, 2, and 3) summarize the migrationdifferences in the stacking gel of each system.Prepare samplesand select buffersSelect the standardChoose the electrophoresischamber system and power supplyRun the gelStain the gelPost stain7Select precast gelHigh-performanceprecast protein gelsIf you are doing standard one-dimensional proteinelectrophoresis, we have a broad range of solutions tofit your research needs. Our selection of precast gelsconsists of several different chemistries, well formats,and gel sizes, so you can get the protein separationyou need for accurate downstream results.Bolt Bis-Tris Plus gel. Learn more at thermofisher.com/proteingelsGLYCINE(Trailing Ion)PROTEIN/SDS COMPLEX(Stacked Proteins)CHLORIDE(Leading Ion)PROGRESSION OF RUNFigure 1. The Tris-glycine gel system. Gel buffer ions are Tris and chloride(pH 8.7) Running buffer ions are Tris, glycine,and SDS (pH 8.3) Gel operating pH is 9.5Common Ion is Tris,present in the gel and running buffersMES or MOPS(Trailing Ion)PROTEIN/SDS COMPLEX(Stacked Proteins)CHLORIDE(Leading Ion)Figure 2. The Bis-Tris gel system. Gel buffer ions are Bis-Tris andchloride (pH 6.4) Running buffer ions are Tris, MES orMOPS, and SDS (pH 7.3) Gel operating pH is 7.0Precast gelsPopular gel chemistriesSpecialty gels NuPAGE Bis-Tris gels Novex Tricine gels NuPAGE Tris-Acetate gels NativePAGE gels Bolt Bis-Tris Plus gels Novex IEF gels Novex Tris-Glycine gels Novex Zymogram gels E-PAGE gelsDid you know?Over 45 years ago,Ulrich K. Laemmli firstpublished SDS-PAGE as amethod for cleavage analysisof structural proteins inbacteriophage T4.PROGRESSION OF RUNCommon Ion is Bis-tris,present in the gelCasting your own gels?TRICINE(Trailing Ion)PROTEIN/SDS COMPLEX(Stacked Proteins)ACETATE(Leading Ion)Figure 3. The Tris-acetate gel system. Gel buffer ions are Tris and acetate(pH 7.0) Running buffer Ions are Tris, tricine,and SDS (pH 8.3) Gel operating pH is 8.1We offer preassembled empty cassettes, buffers,and reagents. Learn more N OF RUNCommon Ion is Tris,present in the gel and running bufferThe combination of a lower-pH gel buffer (pH 6.4) and runningbuffer (pH 7.3–7.7) leads to a significantly lower operating pH(pH 7.0) during electrophoresis, resulting in better sample integrityand gel stability.Precast protein gelsSample preparation andelectrophoresis buffersProtein standardsElectrophoresis chambersystems and power suppliesElectrophoresisrun conditionsProtein gel stains
8Prepare samplesand select buffersSelect precast gelSelect the standardGel selection guideChoose the electrophoresis chambersystem andpowersupplychambersystemandpower supplyRun the gelStain the gelFind the right gel for your research needs based on molecular weight,downstream applications, and throughput requirements.Molecular weightLow molecular weight proteinsand peptides ( 2.5 kDa)High molecular weight proteins( 500 kDa)NovexTricine gelsNuPAGETris-Acetate gelsHigh-sensitivitywestern blottingBroad-range molecularweight separationLow throughputMedium or high throughputApplicationE-PAGE 48-wellor 96-well gelsDownstream applicationsrequiring high protein integrity(e.g., mass spectrometry)Large sample volume forhigh detection sensitivityBolt Bis-TrisPlus gelsNuPAGEBis-Tris gelsNuPAGEBis-Tris gelsNuPAGEBis-Tris gelsBolt Bis-TrisPlus gelsBolt Bis-TrisPlus gelsBolt Bis-TrisPlus gelsAThe most widely used gel system for separatinga broad range of proteins by SDS-PAGE isthe Laemmli system, which uses Tris-glycinegels comprising a stacking gel componentthat helps focus the proteins into sharp bandsat the beginning of the electrophoretic run andthe resolving gel component that separates theproteins based on size. This classic system usesa discontinuous buffer system where the pH andionic strength of the buffer used for running the gel(Tris, pH 8.3) is different from the buffers used in thestacking gel (Tris, pH 6.8) and resolving gel (Tris,pH 8.8). The highly alkaline operating pH of theLaemmli system may cause band distortion, loss ofresolution, or artifact bands.The major causes of poor band resolution with the Laemmlisystem are:NovexTris-Glycine gels H ydrolysis of polyacrylamide at the high pH of the resolvinggel, resulting in a short shelf life of 8 weeks C hemical alterations such as deamination and alkylationof proteins due to the high pH of the resolving gelNative separationMolecular weight1stdimension2nddimensionIsoelectric pointNativePAGE gelsNovex Tris-Glycinegels with native buffersNovex IEF gelsZOOM IPG stripsNuPAGE Bis-Trisgels, 2D wellNovex Tris-Glycinegels, 2D wellNovex Tris-Glycinegels, 2D wellNovex Tris-GlycineZOOM gels, IPG wellNuPAGE Bis-Trisgels, 2D wellNuPAGE Bis-TrisZOOM gels, IPG well Find the right mini gel using our interactive gel selection toolat thermofisher.com/minigelselectionFor ordering information refer to pages 81–87.Protease activityNovex Zymogramgels (casein,blue casein, or gelatinsubstrates)9Choose the right gel chemistry for your research needsBis-Tris chemistry vs.Tris-glycine chemistryDenaturing separationCoomassie dyeor silver stainingProtein gel electrophoresis technical handbookPost stain123456789 10B12345678910Figure 4. Protein separation using (A) a Bolt Bis-Tris Plus gel and (B)another manufacturer’s traditional Tris-glycine gel.Unlike traditional Tris-glycine gels, NuPAGE and Bolt gels are BisTris HCI–buffered (pH 6.4) and have an operating pH of about 7.0.The neutral operating pH of the Bis-Tris systems provides thefollowing advantages over the Laemmli system: Longer shelf life of 8–12 months due to improved gel stability I mproved protein stability during electrophoresis at neutral pHenabling sharper band resolution and accurate results(Moos et al. 1998) C omplete reduction of disulfides under mild heating conditions(70 C for 10 minutes) and absence of cleavage of Asp-Probonds R educed state of the proteins maintained duringelectrophoresis and blotting of the proteins when usingInvitrogen NuPAGE Antioxidant R eoxidation of reduced disulfides from cysteine-containingproteins, as the redox state of the gel is not constant C leavage of Asp-Pro bonds of proteins when heated at100 C in Laemmli sample buffer, pH 5.2Choosing the right gel percentageIn general, the size of the molecule being separated shoulddictate the acrylamide or agarose percentage you choose. Usea lower percentage gel to resolve larger molecules and a higherpercentage gel to resolve smaller ones. The exception to this ruleis when performing isoelectric focusing. Refer to the gel migrationcharts throughout this chapter to find the gel best suited for yourapplication. As a general rule, molecules should migrate throughabout 70% of the length of the gel for the best resolution. Whenprotein molecular weights are wide ranging, or unknown, gradientgels are usually the best choice.Precast protein gelsSample preparation andelectrophoresis buffersProtein standardsChoosing a well formatand gel thicknessWe offer most polyacrylamide gels in a choice of nine different wellformats (17 well, 15 well, 12 well, 10 well, 9 well, 5 well, 1 well,2D/preparative well, or IPG well). Two thicknesses (1.0 mm and1.5 mm) are also available for popular gel types. If loading largesample volumes ( 30 μL), a thicker gel (1.5 mm) with fewer wells(e.g., 5 well) or a Bolt gel with its higher-capacity wedge wellsis more appropriate. When blotting, remember that proteins willtransfer more easily from a 1.0 mm thick gel than from a 1.5 mmthick gel.Electrophoresis chambersystems and power suppliesElectrophoresisrun conditionsProtein gel stains
Select precast gelPrepare samplesand select buffersSelect the standardChoose the electrophoresischamber system and power supplyRun the gelStain the gelProtein gel electrophoresis technical handbookPost stainBolt Bis-Tris Plus mini gelsFigure 7. Bolt Bis-Tris Plus gel migrationchart. Optimal separation range is shown withinthe gray areas.Neutral-pH gel system with a uniquewedge well designInvitrogen Bolt Bis-Tris Plus gels are precastpolyacrylamide gels designed for optimalseparation of a broad molecular weight range ofproteins under denaturing conditions during gelelectrophoresis (Figure 6 and 7). These gels helpdeliver consistent performance with a neutral-pHenvironment to minimize protein degradation. Theunique wedge well design (Figure 5) allows loadingof up to 2x more sample volume than other precastgels. Bolt gels are ideal for western blot transferand analysis along with any other technique whereprotein integrity is crucial.Bolt Bis-Tris Plus gels offer:Specifications Shelf life: 16 months Average run time: 35 minutes Separation range: 0.3–260 kDa Better protein resolution—gels are 10% longer, allowingdetection of more protein bands than standard mini gels High lot-to-lot consistency—coefficient of variation (CV)of only 2% for Rf values (migration)Figure 5. The unique wedge well designof Bolt Bis-Tris Plus gels.“ For one of our projects in the lab, weresolve proteins by electrophoresis todetermine the accumulation of ubiquitinatedproteins following treatment with aproteasome inhibitor. When we resolvedthe ubiquitinated proteins using theTris-glycine gels, we observed a smear.However, when we switched to resolvingthe ubiquitinated proteins using the BoltBis-Tris gels, we were delightfully surprisedto observe individual protein bands in placeof the smear.” —Susan S., University ofPennsylvania, Philadelphia, US P olyacrylamide concentrations: fixed 8%, 10%, and 12%;gradient 4–12% Gel dimensions: 8 x 8 cm (1 mm thick) M aximum sample volume per 12-well gel: 40 μL, or two-thirdsof the sample well volume P reserved protein integrity—neutral-pH formulationminimizes protein modifications Superior band quality and band volume—Invitrogen Novex Bis-Tris Plus chemistry is designed todeliver sharp, straight bands with higher band volume“ The new Bolt system is wonderful. I amstill amazed that I can run a PAGE gel in23 minutes. The entire system is incrediblyuser friendly from the Bolt precast gels withwedged wells for ease of loading to theMini Gel Tank system. The bands producedfrom the westerns were sharp and straight.I would and have highly recommended thissystem to anyone doing protein work.” —Crystal M., Queen’s University,Ontario, Canadats acquired
complexes. Polyacrylamide has a smaller pore size and is ideal for separating most proteins and smaller nucleic acids. Polyacrylamide gel electrophoresis (PAGE) Polyacrylamide gels are generated by the polymerization of acrylamide monomers. These monomers are crosslinked into long chains by the addition of bifunctional compounds such as
7.5.5. If the gel is attached to the longer slotted plate, carefully use the knife to push the gel foot up through the slot so that the gel can be removed from the plate. Using both hands gently lift the gel and transfer the gel to the prepared container containing Milli Q water. 7.5.6. Allow the gel to wash in the for 5 minutes on a rotary .
SDS-Agarose Gel Electrophoresis SDS-agarose gels contained 0.4% (w/v) agarose. The electrophoresis buffer con-tained 0.1 M tris acetate, 0.003 M EDTA, 0.1% (w/v) SDS. The pH was set at 7.9 with pure acetic acid. Samples were put in the SDS-agarose gel and the gels were run in a horizontal electrophoresis system (Mini-Sub Cell GT—7 10 cm (W x
polyacrylamide mini gel system to perform native (non-denaturing) electrophoresis. The near neutral pH 7.5 environment during electrophoresis results in maximum stability of both proteins and gel matrix, providing better band resolution than other gel systems including the traditional Tris-glycine native electrophoresis (Laemmle) system.
Guide The Novexfi Pre-Cast Gel Electrophoresis Guide contains information about the Novexfi Pre-Cast gels and is intended to supplement the Gel Instruction Cards (IM-6000 to IM-6008) supplied with the pre-cast gels. Complete protocols for sample and buffer preparation, electrophoresis conditions, staining, and blotting are provided in this guide.
Power Supply: Gel Electrophoresis Typical Instructions and Guidelines 1. Place the cover on the electrophoresis unit, making sure to have the negative (black) electrode at the well end of the gel. 2. Insert the electrode cords into the proper inputs of the power supply. Set the power supply on 'low' and turn it on. Set the voltage at 90-95 .
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.
agarose gel electrophoresis, the buffer (Tris-glycine-SDS in this case) is a source of electrolytes (ions) that enables current to flow through the gel. Unlike agarose gel electrophoresis, the SDS-PAGE apparatus holds the gel in a vertical position. Polyacrylamide gels are typically very thin
Precast gel systems consist of precast gels and compatible electrophoresis cells, as well as optional related products such as blotting cells, power supplies, gel dryers, reagents, and buffers. Follow the tables below to find the system that's right for you. Bio-Rad offers the widest selection of precast gel systems to meet today's research needs:
