Western Blotting Tools - Fisher Sci
Western Blotting ToolsPublication Quality Westerns in Minuteswith Millipore’s Pre-optimized Products
Zero to Publication QualityWesterns inMinutes!22Millipore’s Western blotting portfoliodelivers the highest quality in the shortesttime, setting a new pace for discovery.One of the greatest challenges in advancing research is obtainingconsistent, quality results. In Western blotting, the most importantfactor in determining the success of experiments is the quality ofresources used, including the protein extraction kit, transfer membraneand reagents. Millipore offers an array of Western blotting productsthat are pre-optimized to work synergistically, providing strong specificsignals and low background to help you quickly produce publicationquality results.Ready, set, publish! At each step in the Western blotting workflow,choose the Millipore product with unique advantages that will driveyour research forward.ProteinExtraction &PreparationGentle proteinextraction kits, pg. 3Rapid proteinisolation withPureProteome magnetic beads, pg. 3Fast, effectiveconcentration withAmicon Ultracentrifugal filters,pg. 33Electrophoresis& TransferHigh protein bindingwith Immobilon membranes, pg. 4Blocking22-minuteimmunodetectionwith SNAP i.d. protein detectionsystem, pg. 6Protein-free Bløk noise-cancellingreagents, pg. 8AntibodyIncubation,WashingSNAP i.d. proteindetection system,pg. 6Optimizedantibodies for theSNAP i.d. detectionsystem, pg. 9DetectionPremixedLuminata WesternHRP substrates forstronger signals,pg. 10
Protein Extraction& PreparationProtein Extraction &Sample PreparationProtein extraction and purification represent the first of many challenges in obtaining intact, active proteins.Millipore’s quality reagents unite superior performance with speed to reduce exposure of proteins to unfavorableconditions, leading to more stable, intact proteins for downstream analysis.Extraction Kits and Protease InhibitorsProtein stability is fundamental to all aspects of protein research, including analysis by Western blotting. Combine our gentle proteinextraction kits with protease inhibitors to obtain stabilized, intact, and active proteins.DescriptionQty/PkCatalogue No.2145Compartment Protein Extraction Kit1 kitTotal Protein Extraction Kit1 kit2140Nuclear Extraction Kit100 assays2900RIPA Lysis Buffer, 10X100 mL20-188Protease Inhibitor Cocktail1 vial20-201Chymostatin100 mgEI6Leupeptin100 mgEI8Pepstatin A100 mgEI10Affinity PurificationPurify your protein with PureProteome magnetic beads or agarose beads. PureProteome magnetic beads ensure fast, effective isolation ofproteins without sample loss and are available in nickel, protein A, or protein G formats.DescriptionQty/PkCatalogue No.LSKMAGH10PureProteome Nickel Magnetic Beads10 mLPureProteome Protein A Magnetic Beads10 mLLSKMAGA10PureProteome Protein G Magnetic Beads10 mLLSKMAGG10Protein A Agarose, fast flow10 mL16-156Protein G Agarose, fast flow10 mL16-266Streptavidin Agarose Conjugate10 mL16-126Buffer Exchange and ConcentrationSimultaneously concentrate and desalt your samples with Amicon Ultra centrifugal filters. Their unparalleled rapid and reproducibleperformance minimizes protein exposure to harsh buffers.DescriptionQty/PkCatalogue No.Amicon Ultra - 0.5 mL Filters*24/pkUFC50102496/pkUFC501096Amicon Ultra - 4 mL Filters*24/pkUFC801024Amicon Ultra - 15 mL *10,000 MWCO. For additional MWCOs, visit www.millipore.com or contact Technical Service.3
ImmobilonWestern Blotting Transfer MembranesThe signal intensity in a Western blot is highly dependent on theprotein’s density after a Western transfer. When aninsufficient amount of protein is bound to themembrane, a strong specific signal can be verydifficult to obtain. For that reason, the quality ofthe Western blotting transfer membraneis essential to obtaining high signalto-noise ratios and clear experimentalresults.The PVDF Immobilon membranes provide highprotein binding capacity resulting in strong signals.Each of the Immobilon membranes has been optimized for aValidated for theSNAP i.d. systemdifferent protein blotting application.Key FeaturesImmobilon P SQ membrane prevents the proteinsfrom blowing through the membrane, increasingprotein SQ BackupMolecular weight standards (lanes 1 and 3) and calf liver lysate (lanes 2 and4) were transferred to Immobilon-P or Immobilon-PSQ membranes. A sheet ofImmobilon-PSQ membrane was placed behind the primary membranes to captureproteins that passed through (lanes 5 and 6 behind Immobilon-P membrane; lanes7 and 8 behind Immobilon-PSQ membrane).4 High protein binding capacity Easy to strip and reprobe High tensile strength and flexibility Highly resistant to organic solvents
Electrophoresis& TransferMultiplex detection usingfluorescent probes on theImmobilon FL membrane1 ng1000 ngActin-tubulin assay on Immobilon-FLmembrane. Rabbit muscle actin (red) wasdetected using rabbit anti-actin secondaryantibodies and QDot 655 goat anti-rabbitsecondary antibodies. Porcine brain tubulin(green) was detected using mouse antitubulin primary and QDot 565 goat antimouse secondary antibodies. Sensitivitiesdown to 1 ng were observed on a Kodak imager. Data provided by Quantum DotCorporation.1000 ng1 ngComparison of Immobilon membrane propertiesImmobilon-Ptransfer membraneImmobilon-PSQtransfer membraneImmobilon-FLtransfer membraneDescriptionOptimized to bind proteins transferredfrom a variety of gel matricesUniform pore structure results insuperior binding of proteins withMW 20 kDaOptimized for PVDFPVDFPVDFPore size0.45 µm0.2 µm0.45 cations Western blotting Binding assays Amino acid analysis N-terminal protein sequencing Dot/slot blotting Glycoprotein visualization Lipopolysaccharide analysis Mass spectrometry Low molecular weightWestern blotting Amino acid analysis Mass spectrometry N-terminal protein sequencing Western blotting Dot/slot blotting FluorescenceimmunodetectionDetection methods Chromogenic Chemiluminescent Radioactive Chromogenic Chemiluminescent Radioactive Fluorescent Chromogenic Chemifluorescent ChemiluminescentProtein binding capacityInsulin: 160 µg/cm2BSA: 215 µg/cm2Goat IgG: 294 µg/cm2Insulin: 262 µg/cm2BSA: 340 µg/cm2Goat IgG: 448 µg/cm2Insulin: 155 µg/cm2BSA: 205 µg/cm2Goat IgG: 300 µg/cm2Ordering InformationDescriptionSizeQty/PkCatalogue No.Immobilon P: PVDF 0.45 µm7 x 8.4 cm50/pkIPVH0785026.5 cm x 3.75 m1 rollIPVH00010Immobilon P : PVDF 0.2 µmSQImmobilon FL: PVDF 0.45 µm7 x 8.4 cm50/pkISEQ0785026.5 cm x 3.75 m1 rollISEQ000107 x 8.4 cm10/pkIPFL0781026.5 cm x 3.75 m1 rollIPFL00010For a complete listing of available Immobilon membranes, visit www.millipore.com/immobilonwestern.5
SNAP i.d. ProteinDetection SystemRapid immunodetection in just 22 minutes!Western blotting has been the life scientist’s workhorse since its introduction in1979, and it remains the gold standard in protein detection. Few improvementswere made to this laborious, time-consuming technique, until the SNAP i.d.protein detection system was introduced in 2008. The SNAP i.d. systemdecreases the immunodetection phase of Western blotting to 22 minutes byusing two mechanisms to favor antibody-antigen interaction:MembraneMembrane1. The SNAP i.d. protocol uses higher antibody(Ab) concentrationssurfacesurfacerelative to traditional methods, driving antibody (Ab)-antigen (Ag)complex formation.[Ab] [Ag][Ab Ag][Ab] [Ag][Ab Ag]2. The SNAP i.d. system uses vacuum suction to pull the antibodies through the membrane, exposing all of the membraneembedded[Ab] target[Ag] proteins[Abto theAg] antibody. This increases the available antigen concentration, driving the equilibrium tofavor antibody-antigen complex formation.[Ab] [Ag]EntrappedEntrappedproteinprotein[Ab Ag]Key roughthroughthethemembranemembrane 22-minute immunodetectionenabling more experimentsin less time Increased antibody-antigen binding Superior washes for ppedEntrappedproteinproteinDuring electrotransfer of proteins from SDS-PAGE gels to membranes, proteinsget trapped within the membrane’s 3-dimensional structure. Traditional Westernblotting relies on the diffusion of the antibody through the membrane to reachentrappedproteins.This is aBlotveryslow and inefficientprocess.The SNAP TraditionalWesternBlotsystem actively pulls antibodies through the membrane, increasing their lyactivelypullspullsto the entrapped proteins and decreasing the required antibody incubation ntsreagentsthroughthroughthethemembranemembrane6
BlockingAntibodyIncubation, WashingComparison of Westerns using the SNAP i.d. system and traditional immunoblottingTraditional Western BlotSNAP i.d. SystemP 1:80,000S 1:50,000P 1:10,000S 1:20,000Detection of TransferrinBlots of a serial dilution of human serum were probed withanti-human transferrin followed by anti-sheep rabbit HRPconjugated IgG secondary antibody (AP147P, Millipore). Blotswere visualized with Luminata Forte Western HRP Substrate(WBLUF0500, Millipore).P 1:20,000S 1:80,000P 1:1,000S 1:10,000Detection of VimentinBlots of a serial dilution of ReNcell CX cell lysates (14,7, 3 µg) were probed with anti-human vimentin (AB1620,Millipore) followed by anti-goat rabbit HRP-conjugated IgGsecondary antibody (AP106P, Millipore). Blots were visualizedwith Luminata Forte Western HRP Substrate (WBLUF0500,Millipore).P 1:1,000S 1:200,000P 1:200S 1:40,000Detection of MAP Kinase 1/2 (Erk 1/2)Blots of a serial dilution of rat liver lysate (6, 3, 1.5 µg) wereprobed with anti-MAP Kinase 1/2 (06-182, Millipore) followedby anti-goat rabbit HRP-conjugated IgG secondary antibody(AP132P, Millipore). Blots were visualized with LuminataForte Western HRP Substrate (WBLUF0500, Millipore).P 1:5,000S 1:10,000P 1:1,000S 1:10,000Detection of AdenovirusBlots of a serial dilution of Adenovirus infected HEK cellswere probed with anti-Adenovirus clone 20/11 (MAB8052,Millipore) followed by anti-goat mouse HRP-conjugated IgGsecondary antibody (AP124P, Millipore). Blots were visualizedwith Luminata Forte Western HRP Substrate (WBLUF0500,Millipore).A.77 kDaB.58 kDaC.44 kDaD.140 kDaConditionsOrdering InformationDescriptionComponentsQty/PkSNAP i.d. Protein Detection SystemSNAP i.d Consumables and AccessoriesCatalogue No.WBAVDBASESingle Blot HolderDouble Blot HolderTriple Blot HolderAntibody Collection TraySNAP i.d. Blot AVDBH03WBAVDABTRWBAVDR0LL7
BlockingBløkNoise-Cancelling ReagentsBløk reagents are a family of uniquely formulated protein-free blocking solutionsthat provide numerous benefits including: Reduced background for chemiluminescent or fluorescent detection. Unique formulation for detection of phosphorylated proteins. More stable diluent for antibodies than milk. Allows chromogenic staining of membranes after immunodetection.Validated for theSNAP i.d. system Engineered for superior reagent flow through SNAP i.d. blot holders.Competitor LCompetitor PNFDMBløk-PO reagent preserves thephosphorylation state of the proteinBløk – POChemiluminescence detection of pERK inEGF-stimulated A431 lysate (10-2.5 µg/lane,12-110, Millipore) using anti-pERKantibody (1:200, 05-797R, Millipore). Bandswere detected using Luminata ForteWestern HRP substrate (WBLUF0500,Millipore). pERK phosphorylated doubletindicated by arrow. NFDM - Non-fat dry milkPost-immunodetection chromogenicstaining of blotsChemiluminescencedetection1 2 34 5 6Blot stainedafter detection1 2 34 5 6Fluorescencedetection1 2 34 5 6Blot stainedafter detection1 2 34 5 6When weak signals were obtained in lanes5 & 6, blots were stained with Coomassieblue following either chemiluminescence(left blots) or fluorescence (right blots)detection to verify equal loading andprotein transfer of all lanes.Lane 1: Molecular weight markerLane 2: A431 Pervanadate (PVD) stimulatedLane 3: A431 control non stimulated(fresh samples)Lane 4: A431 EGFR stimulated(fresh samples)Lane 5: A431 control non stimulated(old samples)Lane 6: A431 EGFR stimulated(old samples)Ordering InformationDescription8Detection MethodQty/PkCatalogue No.Bløk-CH ReagentChemiluminescence detection500 mL/bottleWBAVDCH01Bløk-FL ReagentFluorescence detection500 mL/bottleWBAVDFL01Bløk-PO ReagentPhosphoprotein detection500 mL/bottleWBAVDP001
AntibodyIncubationOptimized Antibodiesfor the SNAP i.d. SystemObtain fast, reproducible results using antibodies optimized for the SNAP i.d. systemTarget ProteinCatalogue No.SNAP i.d. Dilution Factor orAntibody ConcentrationActin, clone C4MAB15011:2000Akt, phosphotyrosine (Tyr450)07-16431:1000ATR09-0701:500BRCA-1 clone BC7005-8421:500CAF1 p60, clone SS-53, 1-12404-15231 µg/mLCatenin, b06-7341:200c-Jun, clone 6E4.405-10765 µg/mLCREB (bZIP transcription factor)06-8631:200CyPA (Cyclophilin A)07-3131:1000Endophilin B1AB105551:500-1:1000FUBP307-7422 µg/mLG9a (BAT8)09-0711 µg/mLGAPDHMAB3741:8000HDAC1109-8270.5 µg/mLHexim 107-9554 µg/mLHLX109-0842 µg/mLIGF2 mRNA-binding protein 207-1034 µg/mLIGF2 mRNA-binding protein 307-1042 µg/mLIKKa07-10071 µg/mLIKKb07-10082-10 µg/mLIRS1, clone 58-10C-3105-784R0.1 µg/mLJunD07-13341:2000LSM14AABE371 µg/mLMAP Kinase 1/2 (Erk-1/2)06-1821:500MYPT1, phosphothreonine (Thr696)ABS450.1 µg/mLNES, NestinAB59221:1000NEFL, 70 kDa clone DA2MAB16151:200P38/SAP-K205-4541:600P 53AB5651:1000PLCg-1, phosphotyrosine (Tyr783)07-21341:1000PLK1, phosphoserine (Ser137)07-13480.5 µg/mLFor a complete listing, visit the SNAP i.d. Antibody Optimization Reference Guide at www.millipore.com/SNAPab.SNAP i.d. PublicationsMihrshahi R, Barclay AN, Brown MH. J Immunol. 2009 Oct 15; 183(8):4879-86.Fujimori K, Ueno T, et al. J Biol Chem. 2010 Mar 19; 285(12):8880-6.Sakane A, Honda K, Sasaki T. Mol Cell Biol. 2010 Feb; 30(4):1077-87.Many more to be found on www.millipore.com/SnapPub.9
Luminata Western HRPSubstratesPremixed for convenience.Formulated for optimal results.The new Luminata Western HRP Substratesare a family of premixed, ready-to-usechemiluminescent reagents for the detectionof HRP-based Westerns. Pour directly ontothe Western membrane without worryingabout pipetting error. The Luminata Classico,Crescendo and Forte substrates cover a broadValidated for theSNAP i.d. systemrange of sensitivities for all detection needs.Key FeaturesLuminata ClassicoLuminata CrescendoLuminata Forte Conveniently premixed to avoidcross contamination of reagents Broad range of sensitivities tocover all detection needs Consistent results with lesspipetting error Stable at 4 C or room temperature Most sensitive detection reagents Compatible with PVDF andDetection of GAPDH in 10 µg (Lane 1), 5 µg (Lane 2), 2.5 µg (Lane 3), and 1.2 µg(Lane 4) of A431 lysate. Blots were treated with the indicated detection reagentand exposed to x-ray film for 5 minutes. Luminata Forte substrate is equivalent toImmobilon Western Chemiluminescent HRP substrates.10nitrocellulose membranesLuminata ClassicoLuminata CrescendoLuminata ForteBenefitPremixed reagent foreveryday detectionneedsPremixed reagent fordetection within the lowpicogram rangePremixed reagent fordetection within thefemtogram rangeDetection limit 6 picograms 1-3 picograms 400 femtogramsStock solution stability1 year at 4 C1 year at 4 C1 year at roomtemperature
DetectionLuminata substrates provide better protein detection.Dilution series of stimulatedA431 lysates (10-0.6 µg) wereresolved by SDS-PAGE thentransferred on to ImmobilonP membrane. Blots wereblocked with Bløk-CH reagentand probed with either antiPP2, anti-STAT1 (05-987,Millipore), or anti-BRCA1(05-842, Millipore) primaryantibody diluted in Bløk-CHreagent. The appropriatesecondary antibodies wereadded to each antibody. Eachblot was visualized with theindicated HRP detectionreagent. The limit ofdetection for each reagent isindicated in left column. Blotswere processed using theSNAP i.d. protein detectionsystem.Protein:PP2 6 picogramsLuminata ClassicoCompetitor PCompetitor GLuminata CrescendoCompetitor PCompetitor BLuminata ForteCompetitor PCompetitor GProtein:STAT1 1-3 picogramsProtein:BRCA1 400 femtogramsOrdering InformationDescriptionQty/PkCatalogue No.Luminata Classico Western HRP Substrates100 mLWBLUC0100Luminata Classico Western HRP Substrates500 mLWBLUC0500Luminata Crescendo Western HRP Substrates100 mLWBLUR0100Luminata Crescendo Western HRP Substrates500 mLWBLUR0500Luminata Forte Western HRP Substrates100 mLWBLUF0100Luminata Forte Western HRP Substrates500 mLWBLUF0500Immobilon Western Chemiluminescent HRP Substrate100 mLWBKLS0100Immobilon Western Chemiluminescent HRP Substrate500 mLWBKLS0500Related ProductsWestern Blot Enhancing ReagentsAvoid repeating your Western blotting experiment. Enhance your signal with the ChemiLucent Plus kit or stripand reprobe your blot with a different antibody using the ReBlot Plus or Blot Restore reagents.DescriptionQty/PkCatalogue No.ChemiLucent Plus Western Blot Enhancing Kit1 kit2650ReBlot Plus Mild Antibody Stripping Solution, 10X50 mL2502ReBlot Plus Strong Antibody Stripping Solution, 10X50 mL2504Blot Restore Membrane Rejuvenation Kit, 10X1 kit252011
Ordering InformationImmobilon Transfer MembranesDescriptionSizeQty/PkCatalogue No.Immobilon P: PVDF 0.45 µm7 x 8.4 cm50/pkIPVH0785026.5 cm x 3.75 m1 rollIPVH00010Immobilon P : PVDF 0.2 µmSQImmobilon FL: PVDF 0.45 µm7 x 8.4 cm50/pkISEQ0785026.5 cm x 3.75 m1 rollISEQ000107 x 8.4 cm10/pkIPFL0781026.5 cm x 3.75 m1 rollIPFL00010SNAP i.d. SystemDescriptionComponentsQty/PkSingle Blot Holder30/pkWBAVDBH01Double Blot Holder30/pkWBAVDBH02Triple Blot Holder20/pkWBAVDBH03Antibody Collection Tray20/pkWBAVDABTRSNAP i.d. Blot Roller1/pkWBAVDR0LLDescriptionDetection MethodQty/PkCatalogue No.Bløk-CH ReagentChemiluminescence Detection500 mL/bottleWBAVDCH01Bløk-FL ReagentFluorescence Detection500 mL/bottleWBAVDFL01Bløk-PO ReagentPhosphoprotein Detection500 mL/bottleWBAVDP001SNAP i.d. Protein Detection SystemCatalogue No.WBAVDBASESNAP i.d Consumables and AccessoriesBløk Noise-Canceling ReagentsLuminata Western HRP SubstratesWestern Blotting Enhancing ReagentsDescriptionQty/PkCatalogue No.DescriptionQty/PkCatalogue No.Luminata Classico WesternHRP Substrate500 mLWBLUC0500ChemiLucent PlusWestern Blot Enhancing Kit1 kit2650Luminata Crescendo WesternHRP Substrate500 mLWBLUR0500ReBlot Plus Mild AntibodyStripping Solution, 10x50 mL2502Luminata Forte Western HRPSubstrate500 mLWBLUF0500ReBlot Plus Strong AntibodyStripping Solution, 10x50 mL2504TO PLACE AN ORDERIn the U.S. and Canada, call toll-free 1 800-Millipore (1-800-645-5476)In Europe, please call Customer Service:France: 0825.045.645 Spain: 901.516.645 Option 1 Germany: 01805.045.645 Italy: 848.845.645 UK: 0870.900.46.45For other countries across Europe and the world, please visit www.millipore.com/offices.For Technical Service, please visit www.millipore.com/techservice.www.millipore.com /WBtoolsMillipore, Advancing Life Science Together, SNAP i.d., Immobilon,Amicon, and Ultracel are registered trademarks of MilliporeCorporation. ReNcell, PureProteome, Luminata, Bløk, ReBlot,ChemiLucent, and the M mark are trademarks of MilliporeCorporation. Qdot is a trademark of Quantum Dot Corporation.Kodak is a registered trademark of Eastman Kodak Company.Lit. No. PB1033EN00 Printed in the USA 03/10LS-SBU-10-02831 2010 Millipore Corporation, Billerica, MA01821 U.S.A. All rights reserved.
secondary antibody (AP106P, Millipore). Blots were visualized with Luminata Forte Western HRP Substrate (WBLUF0500, Millipore). DETECTION OF MAP KINASE 1/2 (ERK 1/2) Blots of a serial dilution of rat liver lysate (6, 3, 1.5 µg) were probed with anti-MAP Kinase 1/2 (06-182, Millipore) followed by anti-goat rabbit HRP-conjugated IgG secondary .
Western Blotting Protocols for Precise Tris-Glycine Gels . Standard blotting procedures may be used with Precise Tris-Glycine Gels. Below are protocols for wet and semi-dry blotting. Wet Blotting Protocol . 1. Cool the transfer buffer to 4 C. 2. Equilibrate gels in the transfer buffer for 5
western blot technology. Fluorescent western blotting provides accurate, quantitative results, stable signals, and the ability to evaluate multiple protein targets on a single blot, which is known as multiplexing. Multiplexing helps make research more efficient and productive. For example, one can visualize a protein of interest simultaneously with
Fluorescent Western Blot Stripping Buffer (Cat. No. PI62299), which is gentle and designed for quickly removing primary and near-infrared (IR) dye-labeled secondary antibodies from western blots. This stripping buffer is for use with low-fluorescence PVDF membranes only. Tips and tricks—fluorescent western blotting
Western Detection Reagents The ultimate detection tool with exceptional performance and value. Immobilon Western Substrates are optimized for high sensitivity and low background over a broad dynamic range, at a considerably lower cost than other chemiluminescent reagents. High signal intensity and low background allow detection to
Protein Research Tools EMD Millipore is a division of Merck KGaA, Darmstadt, Germany. Western blotting is one of the most commonly used techniques in the lab, yet difficulties persist in obtaining consistent, quality results. At EMD Millipore, we've been helping scientists publish
Affinity Chromatography Vol. 1: Antibodies Björkgatan 30 . GE Healthcare Life Sciences Western Blotting Principles and Methods Western Blotting Principles and Methods 28999897 2-D Electrophoresis Principles and Methods GE Healthcare 2-D Electrophoresis using Immobilized pH Gradients
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