NativePAGE Novex Bis-Tris Gel System
user guideNativePAGE Novex Bis-TrisGel SystemA system for native gel electrophoresisCatalog Numbers BN1001BOX, BN1002BOX, BN1003BOX, andBN1004BOXRevision date 19 March 2012Publication Part number 25-0894MAN0000557For Research Use Only. Not intended for any animal or humantherapeutic or diagnostic use.
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ContentsKit Contents and Storage . ivIntroduction . 1Overview . 1NativePAGE Novex Bis-Tris Gel Specifications . 3NativePAGE Gel System . 4Methods . 7Prepare Samples . 7Prepare Running Buffer . 12Perform Electrophoresis . 14Two-Dimensional Native/SDS-PAGE . 19Coomassie Staining of NativePAGE Gels . 22Silver Staining of NativePAGE Gels . 25Western Blotting . 29Expected Results . 32Troubleshooting . 35Appendix. 37Buffer Recipes . 37Accessory Products . 38Technical Support . 40Purchaser Notification . 41References . 42iii
Kit Contents and StorageTypes of ProductsThis manual is shipped with the following products: For ordering information,go to www.lifetechnologies.com/support or contact Technical Support(page 40).ProductShipping andStorageQuantityNativePAGE Novex 3–12% Bis-Tris GelsBox of 10 gelsNativePAGE Novex 4–16% Bis-Tris GelsBox of 10 gelsThe NativePAGE Novex Bis-Tris Gels are shipped on blue ice. Upon receipt,store the gels at 2ºC to 8ºC.Do not freeze NativePAGE Gels.The expiration date is printed on the gel. To obtain the best results, avoid usingexpired gels or improperly stored gels.Product useivFor research use only. Not intended for any animal or human therapeutic ordiagnostic use.
IntroductionOverviewIntroductionThe NativePAGE Novex Bis-Tris Gel system is a near neutral pH, pre-castpolyacrylamide mini gel system to perform native (non-denaturing)electrophoresis. The near neutral pH 7.5 environment during electrophoresisresults in maximum stability of both proteins and gel matrix, providing betterband resolution than other gel systems including the traditional Tris-glycinenative electrophoresis (Laemmle) system. The NativePAGE Novex Bis-TrisGel system provides a sensitive and high-resolution method for analysis ofnative membrane protein complexes, native soluble proteins, molecular massestimations, and assessing the purity of native proteins.NativePAGE GelSystemThe NativePAGE Gel system is based on the Blue Native Polyacrylamide GelElectrophoresis (BN PAGE) technique developed by Schägger and von Jagow(Schägger & von Jagow, 1991) that uses Coomassie G-250 as a charge-shiftmolecule. For details on the NativePAGE Gel system, see page 4.In standard SDS-PAGE, the charge-shift molecule is SDS. The SDS denaturesproteins and binds to proteins conferring a net negative charge allowing theproteins to migrate in one direction towards the anode. The SDS is present in thesample buffer and running buffer.In BN PAGE, the Coomassie G-250 binds to proteins and confers a net negativecharge while maintaining the proteins in their native state without any proteindenaturation. The G-250 is present in the cathode buffer to provide a continuousflow of G-250 into the gel, and is added to samples containing non-ionicdetergent prior to loading the samples onto the gel. The gels do not contain anyG-250.The binding of G-250 to proteins offers the following advantages resulting inhigh-resolution native electrophoresis (Schägger, 2001):Applications Proteins with basic isoelectric points (pI) normally have a net positive chargethat are converted to proteins with a net negative charge, allowing theproteins to migrate in one direction towards the anode. Membrane proteins and proteins with significant surface-exposedhydrophobic area are less prone to aggregation as G-250 binds nonspecifically to hydrophobic sites converting them to negatively charged sites.The NativePAGE Novex Bis-Tris Gel system is suited for: Analyzing native membrane protein complexes or soluble protein complexes Determining the purity of native proteins, and estimating molecular masses ofnative proteins and complexes Performing Two-Dimensional Native/SDS-PAGE to resolve complex samples Analyzing protein complexes purified using NativePure Native ComplexPurification System from Life Technologies (page 38) Performing in-gel or solution activity assaysContinued on next page1
Overview, ContinuedTypes of GelsThe NativePAGE Novex Bis-Tris Gels are available in different acrylamideconcentrations and well formats (see the following table). Gels are available in1.0-mm thickness only.FeatureGel Acrylamide ConcentrationWell FormatBis-Tris Gels3–12% and 4–16%10 and 15 wellsCompatibilityThe size of a NativePAGE Novex Bis-Tris Gel is 10 10 cm (the gel size is8 8 cm). We recommend using the XCell SureLock Mini-Cell (page 37) for theelectrophoresis of NativePAGE Novex Bis-Tris Gels to obtain optimal andconsistent performance.Purpose of theManualThis manual provides the following information:2 An overview of the NativePAGE Electrophoresis System Instructions for preparing samples and running buffer Instructions for performing native gel electrophoresis using the XCell SureLock Mini-Cell Two-Dimensional native/SDS-PAGE protocol Protocols for staining using Coomassie and silver staining Western blotting protocol using the XCell II Blot Module Examples of expected results Troubleshooting
NativePAGE Novex Bis-Tris Gel SpecificationsSpecificationsLoading VolumesGel Matrix:Acrylamide/BisacrylamideGel Thickness:1.0 mmGel Size:8 cm 8 cmCassette Size:10 cm 10 cmCassette Material:Styrene Copolymer (recycle code 7)Sample Well Configuration:10- and 15-wellThe recommended loading volumes and protein load per band by the detectionmethod are provided in the following table.Well Types10 WellRecommendedMaximum LoadVolume25 μL10 well15 Well15 well15 μLMaximum Protein Load Per Band byDetection g1.0 μg/band Scale yoursample load forthe sensitivity0.5 μg/band of your silverstaining kit.Scale yoursample loadaccording tothesensitivityFor use with the of yourSilverQuest or detectionSilverXpress method.Silver StainingKits, werecommend aprotein load of 50 ng/band.3
NativePAGE Gel SystemIntroductionThe ability to maintain native protein conformation and provide highresolution native electrophoresis makes the NativePAGE Bis-Tris Gel System apowerful system for analyzing native protein complexes as compared totraditional native electrophoresis systems such as the Tris-Glycine system(Schägger et al., 1994).The traditional Tris-Glycine (Laemmle) gel system is the most widely usednative electrophoresis system but offers the following limitations: The high operative pH of the Tris-Glycine system adversely affects someproteins that are sensitive to high pH conditions It is incompatible with native samples that require a non-ionic detergent forprotein solubilizationSystem components and general information on the NativePAGE Gel systemare included in this section. For system overview, see page 1.SystemComponentsNativePAGE Novex Bis-TrisGelsThe NativePAGE Novex Bis-Tris Gel System consists of: NativePAGE Novex Bis-Tris [Bis (2-hydroxyethyl) imino-tris(hydroxymethyl) methane-HCl] Mini Gels for separating proteins andprotein complexes NativePAGE Sample Buffer (4X) and NativePAGE 5% G-250 SampleAdditive for sample preparation NativePAGE Running Buffer (20X) and NativePAGE Cathode BufferAdditive (20X) for native electrophoresis NuPAGE Transfer Buffer for blotting of NativePAGE Novex Bis-TrisGelsThe NativePAGE Novex Bis-Tris Gel is a 1.0 mm thick, 8 x 8 cm mini gel usedfor native (non-denaturing) gel electrophoresis of protein samples.The NativePAGE Novex Bis-Tris Gels are used with NativePAGE RunningBuffers (see page 5) to produce a non-denaturing electrophoresis systemoperating at near neutral pH. The near neutral pH environment duringelectrophoresis results in maximum stability of both proteins and gel matrix,providing better band resolution than other gel systems.Continued on next page4
NativePAGE Gel System, ContinuedEstimating SizeThe use of G-250 charge-shift in NativePAGE gels results in protein resolutionbased upon protein size allowing accurate size estimation of native proteinsand protein complexes (Schägger et al., 1994).However, since the proteins maintain their native conformation, the sizeestimation may have an expected size estimation error of 15%. For example, ifyou estimated the molecular mass of a protein to be 450 kDa usingNativePAGE gels, the actual mass may vary between 380-520 kDa. Due to thelarge diversity of protein structure and characteristics, we recommendverifying the molecular mass of native proteins using other techniques such asgel filtration or mass spectrometry.Differences in size estimations using NativePAGE gels are produced by slowmigration and overestimation of mass which can arise due to:NativePAGE BisTris Buffer SystemAdvantages Non-ideal binding of G-250 produces an incomplete or absent charge-shift Protein structures that significantly deviate from globularity or that haveopen interior space have a size, or diameter, that is unusually large for theirmass Proteins with acidic pI’s and compact structures may migrate faster orglycosylated proteins may migrate slower and resolve into diffuse bandsdue to heterogeneity in glycosylation Proteins that bind lipids may migrate at different rates when prepared withdifferent concentrations of detergent due to variation in the amount lipidremaining on the protein at different detergent concentrations.The NativePAGE Bis-Tris non-denaturing buffer system involves three ions: Chloride (-) is supplied by the gel buffer and serves as a leading ion due toits ion mobility as compared to other anions in the system. The gel bufferions are BisTris ( ) and Cl- (pH 6.8). Tricine (-) serves as the trailing ion. The running buffer ions are BisTris andTricine (pH 6.8 BisTris ( ) is the common ion present in the gel buffer and running buffer.During electrophoresis, the operative pH is 7.5.The operating near neutral pH of NativePAGE Novex Bis-Tris Gels andbuffers provide the following advantages over the Tris-Glycine (Laemmle) Gelsystem: Longer shelf life of up to 6 months due to improved gel stability Allows the protein to retain the native structure and activity asdemonstrated by in-gel and in solution activity of proteins afterNativePAGE electrophoresis (Schägger & von Jagow, 1991; Zerbetto et al.,1997) Improved protein stability during electrophoresis at near neutral pHresulting in sharper band resolution and accurate resultsContinued on next page5
NativePAGE Gel System, ContinuedSeparation RangeThe NativePAGE Novex Bis-Tris Gels have a wide range of separationthroughout the low and high molecular weight ranges.The NativePAGE Novex 3–12% Bis-Tris Gels resolve proteins in themolecular weight range of 30-10,000 kDa.The NativePAGE Novex 4–16% Bis-Tris Gels resolve proteins in themolecular weight range of 15-1,000 kDa.To choose the correct NativePAGE Novex Bis-Tris Gel for your application,refer to tionsThe NativePAGE Novex Bis-Tris Gels are compatible with most stainingprotocols including silver, and Coomassie stains.The SilverQuest Silver Staining Kit or SilverXpress Silver Staining Kit(page 25) is suitable for silver staining of NativePAGE Gels. For best resultsand better background, we recommend using the SilverQuest Silver StainingKit.The NativePAGE Novex Bis-Tris Gels are compatible with any of thestandard Coomassie staining procedures. The Novex Colloidal Blue StainingKit (page 22) is recommended for staining NativePAGE Gels.For Western blotting applications, we recommend using a semi-wet transferapparatus such as the XCell II Blot Module (page 29) to blot NativePAGE Gels.6
MethodsPrepare SamplesImportantDue to the large diversity of proteins present in different cells and tissues, it isnot possible to offer a sample preparation protocol that is suitable for allproteins. Based on the starting material and goal of the experiment, the samplepreparation protocol needs to be determined empirically.Brief procedures for sample preparation are described on the following pages.You may use this procedure as a starting point for your lysate and thenoptimize the procedure based on the initial results.Objectives ofSamplePreparationNativePAGE Sample Prep KitThe major objectives of sample preparation are to: Completely solubilize the proteins Maintain proteins in solution in their native state during electrophoresis Prevent protein modifications and proteolysisThe NativePAGE Sample Prep Kit (page 38) includes sample preparationreagents for native gel electrophoresis. The kit includes ready-to-use detergentsolutions (10% DDM and 5% Digitonin) that improve the solubility ofhydrophobic and membrane proteins during sample preparation.The samples prepared with 10% DDM (n-dodecyl-β-D-maltoside),5% Digitonin, or the NativePAGE Sample Prep Kit are compatible withNativePAGE Novex Bis-Tris Gels for native gel electrophoresis showingincreased resolution and reduced streaking.NativePAGE 5%G-250 SampleAdditiveThe NativePAGE 5% G-250 Sample Additive is a concentrated stock solutionof Coomassie G-250 designed for use with detergent (non-ionic) containingsamples prepared for NativePAGE gel electrophoresis.The G-250 dye displaces detergent or loosely bound lipid molecules frommembrane proteins and protein complexes prepared in native bufferscontaining non-ionic detergents, converting hydrophobic sites to negativelycharged sites required for NativePAGE electrophoresis (see page 1 for details).This prevents membrane proteins from aggregating during separation on aNativePAGE gel which does not contain any solubilizing detergent. The G-250dye also binds to detergent molecules in the sample and carries them in thedye-front, ahead of resolving proteins to minimize vertical streaking.The NativePAGE 5% G-250 Sample Additive is added to detergent containingsamples just prior to loading samples onto a NativePAGE gel such that thefinal G-250 concentration in the sample is 1/4th to 1/10th of the detergentconcentration (Schägger, 2001).Continued on next page7
Prepare Samples, ContinuedUse the NativePAGE Sample Buffer (4X) to prepare samples for native (nonNativePAGE Sample Buffer (4X) denaturing) gel electrophoresis with the NativePAGE Novex Bis-Tris Gels.The NativePAGE Sample Buffer (4X) is formulated for native gel electrophoresisand contains BisTris buffer, pH 7.2, NaCl, glycerol, and Ponceau S.General Guidelines NativeMark Unstained ProteinStandardSolubilize the proteins or protein complexes using the minimum amount ofdetergent necessary for maximal solubilization. Maintain the samples on ice during sample preparation and do not heatsamples prior to electrophoresis. You may need to prepare your protein samples with 10% DDM (page 38),5% Digitonin (page 37), or other detergents to determine the best solubilizerfor your protein (Eubel et al., 2005; Schägger, 2001). Maintain the salt concentration of the sample at 50 mM. For detergent containing samples, always add NativePAGE 5% G-250Sample Additive prior to loading samples onto the gel. For detergent-free samples, addition of NativePAGE 5% G-250 SampleAdditive is optional. Prepare samples in 1X NativePAGE Sample Buffer, if possible. If your sample is in a SDS-PAGE sample buffer, prepare a fresh lysatewithout SDS using the detergents included in the sample prep kit. Do notuse SDS-PAGE samples for native gel electrophoresis. You may add protease inhibitors in your sample preparation. Variousprotease inhibitor cocktails are commercially available. Avoid using a complex sample preparation strategy as it may result inprotein loss. If a precipitate forms in the 5% Digitonin solution, heat the solution at 95 Cfor 5 minutes and vortex slowly to dissolve the precipitate. Cool to roomtemperature prior to use. The 5% Digitonin will stay in solution at roomtemperature for up to a week. You may reheat the solution multiple timeswithout any loss in activity. Always wear gloves, protective eyewear, and a laboratory coat whilehandling the detergents. Digitonin is toxic and handle with care, avoid anyexposure of Digitonin to skin.NativeMark Unstained Protein Standard (page 38) is specifically designed foruse with NativePAGE Novex Bis-Tris Gels and consists of 8 protein bands thatallow accurate molecular weight estimation in the range of 20–1200 kDa. Thestandard is supplied in a ready-to-use format and is easily visualized withCoomassie or silver staining, and also with membrane stains such as Ponceau S,or Coomassie after western transfer.Continued on next page8
Prepare Samples, ContinuedMaterials NeededYou will need the following items. See page 38 for ordering information. Protein sample NativeMark Unstained Protein Standard NativePAGE Sample Buffer (4X) Deionized water Homogenization unit for tissue samples Optional: Protease inhibitor cocktail and Benzonase nuclease (Sigma, cat. no.E-1014)For samples that need detergent solubilization:PrepareCell/TissueLysates NativePAGE Sample Prep Kit, 10% DDM (n-dodecyl-β-D-maltoside), or5% Digitonin NativePAGE 5% G-250 Sample AdditiveUse
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.
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Eingangswechselspannung 100 V bis 240 V AC Frequenz 50/60 Hz, einphasig Betriebsumgebung Temperatur: 0 bis 40 C (32 F bis 104 F) Relative Luftfeuchtigkeit: 8 bis 80 % RH Lagerung Temperatur: -20 bis 60 C (-5 F bis 140 F) Relative Luftfeuchtigkeit: 5 bis 95 % RH Maximale Betriebshöhe 5.000 m (16.400 ft)
Eingangswechselspannung 100 V bis 240 V AC Frequenz 50/60 Hz, einphasig Betriebsumgebung Temperatur: 0 bis 35 C (32 F bis 95 F) Relative Luftfeuchtigkeit: 8 bis 80 % RH Lagerung Temperatur: -20 bis 60 C (-5 F bis 140 F) Relative Luftfeuchtigkeit: 5 bis 95 % RH Maximale Betriebshöhe 5.000 m (16.400 ft)
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 .
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