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Focus Western Blot

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November 2018
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Focus Western Blot AH diagnostics: Your Science – Our Focus Dedicated to helping life science and diagnostics laboratories perform their best by providing innovative, high-quality products and expert knowledge. Representing more than 35 suppliers, our application specialists are ready to help you with your next project, from assay setup, training and implementation to analysis. We offer the most cutting edge technologies within ELISA, flow cytometry, imaging, pathology, protein analysis, qPCR, single-cell analysis and bio appliance. We have 30 years of experience serving the Nordic Region and offices in Copenhagen, Aarhus, Oslo, Stockholm and Helsinki. Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY Western Blot Overview Western blotting is as a simple and straightforward technique for protein characterization. However, a great number of steps are required to secure optimal results, and we know it is crucial to be confident with the entire process. We have therefore published this magazine to support you in making the right Western blotting choice. Use the above workflow throughout the magazine as a guideline in your daily Western blot work. Simply identify your step of interest and become inspired by our solutions. Despite Western blotting being a widely used and simple technique, many challenges can still arise. If you come across a problem, turn to the troubleshooting section at the end of the magazine, or contact one of our trained specialists for further support. 1 Sample Preparation�� 5 1.1 Cell Lysis�� 5 1.2 Protease and Phosphatase Inhibition �� 6 1.3 Purification�� 7 1.3.1 His-Tag purification�� 7 1.3.2 FLAG-Tag Purification products�� 8 1.3.3 GST-Tag Purification products�� 9 1.3.4 Myc-Tagged Protein Purification�� 9 1.3.5 Non-Tagged protein purification��10 1.4 Concentration Measuring��12 2 Protein Separation�� 13 2.1 Protein Concentration and Buffer Exchange��13 2.2 Sample Loading Buffers��14 2.2.1 Reducing and Non-reducing Sample Loading Buffers��14 2.2.2 Real-time, In-gel Protein Stain��14 2.3 Gel Electrophoresis Buffers��15 2.4 Molecular Weight Standards��15 2.5 Gel Cast and Run��16 2.6 Protein Stain��16 3 Blotting�� 17 3.1 Membranes��17 3.2 Blotting Papers��18 3.3 Transfer Buffer��18 3.4 Protein Stain for Membranes��19 4 Incubation with Antibodies�� 20 4.1 Blocking Solution��20 4.2 Washing Buffer for Western Blots��20 4.3 Direct or Indirect Methods?��20 4.4 Primary Antibodies��21 4.5 Secondary Antibodies��21 4.6 Streptavidin Conjugates��23 5 Detection�� 24 5.1 HRP Substrates��24 5.2 How to Choose the Right Chemistry for Your Western Blot ��25 5.2.1 Chemiluminescence��25 5.2.2 Fluorescence��25 6 Troubleshooting�� 27 6.1 Unusual/Unexpected Bands��27 6.2 No Bands, Faint Bands or Weak Signal��28 6.3 Electrophoretic Problems��28 6.4 High Background��29 3 4 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 1 Sample Preparation The first step in obtaining a successful Western blot is to properly prepare the starting material. Prior to sample preparation, it is of the utmost importance to acquire knowledge about the protein location, protein source, hydrophobicity of protein target, etc. Whether you are working with cultured cell lines or tissue samples, sample preparation is often a process of trial and error in which you need to choose the right lysis buffers, enzyme inhibitors and appropriate volumes. Some samples may even require mechanical disruption. 1.1 Cell Lysis Cell lysis is often performed using a buffer containing detergents and/or denaturants. The choice of buffer depends on cell location. From Santa Cruz, we offer: Protein location Buffer Whole cell NP-40, RIPA, Triton X-100 Cytoplasmic (soluble) Tris-HCl Cytoplasmic (cytoskeletal bound) Tris-Triton Nuclear, mitochondrial, membrane RIPA, Triton X-100, NP-40 Clontech offers the mild, non-denaturing xTractor Lysis Buffer for fast and easy extraction of proteins from bacteria, yeast, mammalia, and baculo-infected cells. The product is compatible with IMAC resins for quick purification of His-tagged proteins. It takes only 10 minutes! Cat. no. Description Size 635625 xTractor Buffer 500 mL 635656 xTractor Buffer 100 mL 635671 xTractor Buffer 250 mL 635623 xTractor Buffer Kit 10 extractions 5 ► Sample Preparation ► Protein Separation Blotting ► AB Incubation ► Detection YY 1.2 Protease and Phosphatase Inhibition Cell lysis is associated with the release of proteases and phosphatases, which in turn negatively affects the Western blot. UltraCruz® Protease Inhibitor Cocktail Tablet prevents proteolysis from bacterial, yeast, mammalian and plant extracts! Available with or without EDTA. Simply dissolve the tablet in buffer or lysate. Cat. no. Description Size Sc-29130 UltraCruz Protease Inhibitor Cocktail Tablet 20 tablets Sc-29131 UltraCruz® Protease Inhibitor Cocktail Tablet, EDTA-free 20 tablets ® ProteoGuard™ EDTA-Free Protease Inhibitor from Clontech is a cocktail in solution. Provided in single-use tubes for ease of use. Cat. no. Description Size 635672 ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail 100 uL 635673 ProteoGuard™ EDTA-Free Protease Inhibitor Cocktail 10 x 100 uL For phosphatase inhibitors, use the following mixes from Santa Cruz: Cat. no. Size Inhibitor mix Target Sc-45044 1 mL Phosphatase Inhibitor Cocktail A Serine/threonine protein phosphatases, L-isozymes of alkaline Sc-45045 1 vial* Phosphatase Inhibitor Cocktail B Tyrosine phosphatases, acid and alkaline phosphatases Sc-45065 1 mL Phosphatase Inhibitor Cocktail C Alkaline phosphatases, serine/ threonine phosphatases *reconstitute in 1 mL water 6 Looking for inhibitors that target specific groups of proteases and phosphatases? Contact us for customized solutions from Santa Cruz or Enzo. Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 1.3 Purification It can be advantageous to purify proteins by other means than lysing the cells with detergents and denaturants combined with mechanical stress and inhibitors. A large number of methods are available both for purifications prior to Western blotting studies, but also for larger scale downstream purifications. Choose your protein purification products from Takara Clontech. Whether you are working with tagged or non-tagged targets,Takara Clontech offers products for a large number of applications. The below listed categories contains their full protein purification product range. His-tag purification products • Capturem™ His-tagged Purification Kit • His60 Ni • TALON® • Magnetic Beads FLAG-tag purification products • Magnetic DYKDDDDK Immunoprecipitation Kit • Anti-DYKDDDDK Beads GST-tag purification products • Glutathione-Superflow Resin Phosphoprotein enrichment products • Phosphoprotein Enrichment Kit • TALON® PMAC Magnetic Phospho Enrichment Kit • Phosphopeptide Enrichment Spin Columns Glycoprotein enrichment products • Glycoprotein Enrichment Resin Antibody purification products • Thiophilic-Superflow Resin Myc-tag purification products • c-Myc Monoclonal Antibody-Agarose Beads • Magnetic Myc Immunoprecipitation Kit Please contact us for ordering information and further details on the specific products. 1.3.1 His-Tag purification Many conventional methods for protein purification require a lot of time and effort. Immobilized metal affinity chromatography resin columns provide high capacity, but speed and ease of use are compromised due to long equilibration and binding times, as well as slow diffusion of large molecules through the resin bed. A next-generation membrane technology utilizing spin columns, which overcomes these bottlenecks, is now available from Takara Clontech. Purify His-tagged proteins in 5-15 minutes with Capturem® His-tagged purification kit • • • • NEW! 4 step protocol at room temperature Purification of mammalian or bacterial His-tagged proteins Compatible with both native and denaturing conditions containing DTT, β-mercaptoethanol, TCEP, EDTA or glycerol High yield and purity 7 Sample Preparation ► Protein Separation ► ► Blotting AB Incubation YY Equilibration: Add xTractor buffer, spin, discard flowthrough Binding: Load cleared lysate, spin + save flowthrough, transfer column to new tube Washing: Add wash buffer. If needed, add small amount of elution buffer to change imidazole conc. and optimize, spin, save flowthrough, transfer column to new tube Elution: Add elution buffer, spin, elution contains purified and tagged protein Miniprep Maxiprep High-throughput 96 minipreps Cat. no. 635710 635713 635714 Yield ~ 100 µg/prep ~ 2.5 mg/prep ~ 100 µg/prep Sample volume from overnight culture 2-5 mL 10-150 mL 2-5 mL Preparation time 5 min 15 min Concentration of eluted protein ~ 0.3-1 mg/mL Up to 4.5 mg/mL ~ 0.3-1 mg/mL Number of purifications 20 reactions 6 preps 96 preps Use the same buffers for all purification scales – seamless transition Capturem Miniprep ► Capturem Maxiprep ► Talon/His60 1.3.2 FLAG-Tag Purification products • Ideal for identifying and monitoring expression of DYKDDDDK- or FLAG-tagged proteins • Highly specific antibody does not cross-react with endogenous proteins • Use beads to purify or immunoprecipitate FLAG-tagged fusion proteins from cell lysates 8 ► Detection Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY Cat.no. Description Size 635686 Anti- DYKDDDDK Beads 1 mL 635687 Immunoprecipitation Buffer Set 39 rxns 635694 Magnetic DYKDDDDK Immunoprecipitation Kit 50 rxns 635695 Anti-DYKDDDDK Magnetic Beads Each 635696 Magnetic Beads Immunoprecipitation Buffer Set 50 rxns 1.3.3 GST-Tag Purification products • • • • • Easy to use: No optimization required Purifies more protein: High binding capacity (>10 mg of recombinant GST fusion protein per mL resin) Yields highly purified protein: Eluted protein is free of cellular contaminants Reusable: Easy protocol for regenerating and storing resin Provided in flexible formats: • Bulk resin suitable for batch/gravity-flow purification and FPLC applications • Prepacked gravity-flow columns • Ready-to-use purification kit (5 columns and sufficient reagents for 5 purifications) Cat.no. Description Size 635607 Glutathione-Superflow Resin 10 mL 635608 Glutathione-Superflow Resin 100 mL 635619 GST Purification Kit 5 rxns 1.3.4 Myc-Tagged Protein Purification Monoclonal c-Myc Antibody crosslinked to immobilized protein A agarose or magnetic beads. • Provides quick and easy separation of myc-tagged proteins • Yields highly pure denatured proteins • Highly specific antibody to myc, does not cross-react with endogenous proteins Cat.no. Description Size 631208 c-Myc Monoclonal Antibody-Agarose Beads 1 mL 635698 Magnetic Myc Immunoprecipitation Kit 50 rxns 9 Sample Preparation ► Protein Separation ► ► Blotting AB Incubation ► Detection YY 1.3.5 Non-Tagged protein purification Although protein tagging enables simple, effective affinity purification of a wide variety of proteins, there are a number of reasons why it is not necessarily the best method for every protein: • Tags cannot be used to purify endogenous proteins; they can only be used to purify proteins that have been genetically manipulated, which may not be possible or desirable. • In some cases, a tag may interfere with the structure or biological activity of a target protein, or may be difficult to remove without altering its structure or function. • Protein tagging may be unnecessary for proteins with endogenous post-translational modifications or affinity binding sites, such as phosphoproteins, glycoproteins, or antibodies, which can bind specifically to alternative affinity resins. Phosphoprotein Enrichment Phosphoprotein enrichment with a unique IMAC (immobilized metal affinity chromatography) resin provides an effective affinitybased procedure for isolating phosphorylated proteins from mammalian cells and tissues, using gravity columns or magnetic beads. A similar IMAC resin-based method is available for isolating phosphopeptides, using spin columns or magnetic beads. Add Extraction/ Loading Buffer (Buffer A) to cell pellet Total cellular protein Phosphoprotein Affinity Column Resin Flowthrough Mix, incubate, and centrifuge Cell pellet Load sample Was and elute with Buffer B Phosporylated proteins Cat.no. Description Size 635643 Magnetic Phosphopeptide Enrichment Kit 300 rxns 635635 Phosphopeptide Enrichment Buffer Kit Each 635634 Phosphopeptide Enrichment Spin Columns Each 635624 Phosphoprotein Enrichment Kit 6 preps 635641 TALON PMAC Magnetic Phospho Enrichment Kit Each 10 ® Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY Glycoprotein Enrichment Glycoprotein enrichment with a boronic acid-based resin provides quick, efficient, and specific enrichment of glycoproteins from complex samples such as human serum, urine, and spinal fluid, using either simple gravity flow columns or medium pressure methods such as FPLC. Enriched glycoproteins transferred to membranes for Western blotting are selectively detected using Glycoprotein Western Detection Kit. It provides highly sensitive and rapid detection—yielding results within one hour. Cat.no. Description Size 635647 Glycoprotein Enrichment Resin 10 mL 635648 Glycoprotein Western Detection Kit 20 rxns Antibody Purification Antibody purification using thiophilic resins is a simple, powerful, and economical alternative to Protein A, the most common method of immunoglobulin purification. Although Protein A antibody purification is very common, there are certain types of antibodies, such as the single-chain antibodies IgE, IgY, and IgM, that cannot be purified using Protein A. An alternative antibody purification method, thiophilic affinity chromatography (TAC), is ideal for these types of applications, as well as immunoglobulin purification in general, including IgG, IgM, IgA, Fab and Fc fragments, and C3 and C4 complement factors. Thiophilic Resin Add salt Filter/ Centrifuge Load Wash Elute in neutral buffer (pH 7.0) 2 days Protein A Filter/ Centrifuge Load Wash Cat.no Description Size 635616 Thiophilic-Superflow Resin 10 mL 635617 Thiophilic-Superflow Resin 100 mL 635614 Thiophilic-Uniflow Resin 100 mL Elute in low pH buffer (pH ≤ 3.0) Perform dialysis Concentrate protein 11 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 1.4 Concentration Measuring After extracting the proteins from the samples and prior to SDS-PAGE, it is important to determine the total protein concentration in each sample. By measuring the concentration, it is possible to ensure the correct amount of protein to be loaded on the gel, thereby minimizing high background and nonspecific antibody binding. Concentration measurements are also useful for semi-quantitative Western blotting – simply load the same amount of protein in each lane and compare the relative levels of the target protein. DeNovix is a new and innovative instrument for measuring concentrations in an easy and intuitive way. Rapidly measure protein samples before proceeding to Western blot analysis. A 0.5 – 1.0 µL sample is all that is required DeNovix advantages for protein studies: • • • • • • • Fluorometer and spectrophotometer in one instrument! Protein (A280 and labeled) and peptide (A205/215) analysis High absorbance capabilities: up 1125 mg/mL BSA Compatible with Qubit and Quant-it fluorescence assays Built-in cuvette heater controls temperatures for kinetic studies Small footprint: 20 cm x 33 cm Weight: 2 kg Also ideal for quantifying DNA and RNA samples! Fluorometer: 0.5 mL thin-walled PCR tube Cuvette: Standard quartz and disposable cuvettes, full spectrum UV-Vis Microvolume: 0.5 – 1.0 µL, full spectrum UV-Vis Cat. no. DS-11 FX + √ DS-11 FX √ DS-11 + √ DS-11 √ QFX FLUOROMETER 12 √ √ √ √ √ Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 2 Protein Separation After sample preparation, polyacrylamide gel electrophoresis (PAGE) is carried out. Most often, the samples are treated with the negatively charged SDS to unfold and separate the protein content by size. It is also possible to perform non-reducing gel electrophoresis to maintain the native state of the protein. Furthermore, it is helpful to consider the polyacrylamide gel percentage, which is dependent on protein size – larger proteins needs lower acrylamide concentration and vice versa. 2.1 Protein Concentration and Buffer Exchange Prior to protein separation, it is often necessary to concentrate proteins and remove contents, which interferes with gel electrophoresis. The Afyon SDS-PAGE Sample Preparation Kit removes unwanted buffer contents and concentrates protein samples to save you hours in the lab. Superior advantages: • • • • • Contaminants (GuHCl, urea, ammonium, sulfate etc.) are efficiently removed It takes less than 10 minutes Normalization of protein concentration – final filtrate contains exactly 5 µg Non-toxic Compatible with SDS-PAGE as well as chemiluminescent and fluorescent Western blotting Afyon protocol in just 10 minutes: Extract sample ► Add 20 µL Afyon Resin to sample*, vortex, centrifuge, remove supernatant (< 5 min) ► Suspend pellet in loading buffer, centrifuge with spin filter (< 5 min) ► Filtrate contains exactly 5 µg to load directly on gel ► Run gel *Compatible with dilute samples and samples solubilized in GuHCI, detergents, etc. Cat. no. Description Size K-02101-010 Afyon SDS-PAGE Sample Preparation Kit 10 reactions K-02101-025 Afyon SDS-PAGE Sample Preparation Kit 25 reactions 13 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 2.2 Sample Loading Buffers Most often, the prepared samples can be stored at -20°C until use. Prior to gel electrophoresis, the samples are prepared with either a reducing or non-reducing loading buffer, which ensures the right viscosity, color for monitoring the loading and running, denaturation, reduction and pH maintenance. 2.2.1 Reducing and Non-reducing Sample Loading Buffers Avoid wasting time and use pre-mixed loading buffers for SDS-PAGE. Simply add your sample to the loading buffer, boil and load. Samples can remain at room temperature for immediate use, or they can be stored at 4°C or -20°C for later analysis. Choose between reducing and non-reducing loading buffers – both are compatible with staining and Western blotting applications. Cat. no. Description Volume R-03018-B10 Non-reducing protein sample loading buffer (2x) 1 mL R-03018-B50 Non-reducing protein sample loading buffer (2x) 5 mL R-03019-B10 Reducing protein sample loading buffer (2x) 1 mL R-03019-B50 Reducing protein sample loading buffer (2x) 5 mL 2.2.2 Real-time, In-gel Protein Stain After gel electrophoresis, it is important to visualize your protein bands to ensure that the separation is successful. This is known to be a time-consuming and inconvenient process. Visio in-gel protein stain from Advansta helps you to quickly and easily detect protein bands while running your gel. • • • • Instant results: follow the visible bands while running the gel Sensitive: detect down to 10 ng per band Fast: heat your samples with Visio for 10 minutes and go Compatible: use with both reducing and non-reducing gels. Analyze your results with downstream applications* Cat. no. Description Volume K-11053-300 Visio Real-Time Protein Stain, 100 lanes or 10 gels 0.3 mL K-11053-B30 Visio Real-Time Protein Stain, 1,000 lanes or 100 gels 3 mL *Compatible with mass spectrometry. The product has not been validated with downstream Western blotting, but many customers have had success with it. 14 1) Mix Visio with protein sample 2) Incubation at 98 °C for 10 min, vortex and centrifuge briefly – add reducing buffer for reducing gels 3) Load 4) Run Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 2.3 Gel Electrophoresis Buffers To ensure the right pH, electrical conductivity and a negative charge on the proteins, the right composition of running buffer should be used. Save time with Avant Buffer Pouches. By adding just 500 mL of deionized water and mixing, you will have a freshly made, ultrapure buffer for your SDS-PAGE, which ensures reproducible results. Cat. no. Description Size R-01038-020 PBS (concentration 1x at 500 mL) 20 pouches R-01039-020 TBS (concentration 1x at 500 mL) 20 pouches R-01037-020 Tris-Glycine PAGE running buffer (concentration 1x at 500 mL) 20 pouches R-01036-020 Tris-Glycine SDS-PAGE running buffer (concentration 1x at 500 mL) 20 pouches 2.4 Molecular Weight Standards In addition to the Western blot signal, knowledge of the protein size is very useful, which can be confirmed with molecular weight standards. From Santa Cruz, we offer a number of standards, which are provided in SDS-PAGE loading buffer for direct use on your gel: • • Available in low, broad and high range. Use Cruz Marker™ Molecular Weight Standards for visualization on the final Western blot after incubation with the Cruz Marker™-compatible secondary antibodies for Western blotting. Choose between either HRP or AP conjugated secondary antibodies. Cat. no. Description Size Sc-2035 Cruz Marker™ Molecular Weight Standards sufficient for 50 gels Sc-2361 Broad Range Markers 500 µL Sc-2362 High Range Markers 500 µL Sc-2360 Low Range Markers 500 µL 15 Sample Preparation ► ► Protein Separation Blotting ► AB Incubation ► Detection YY 2.5 Gel Cast and Run Electrophoresis performance is dependent on a uniformly cast gel and a high-quality running apparatus. UltraCruz® Electrophoresis Cell – simple, safe and easy • • • • Two-in-one cell suitable for both gel casting and run. Can run up to 10 or 15 samples. Power turns off automatically when lid is opened High resolution separation Cat. no Description Sc-201625 UltraCruz® Electrophoresis Cell 2.6 Protein Stain Check the quality of protein resolution on your gel or the protein transfer on your membrane by staining. Visualize total protein on your gel or blot with the non-toxic fluorescent AdvanStain Scarlet to detect down to 1 ng on laser or CCD imaging systems. Destain your gel and you are ready for downstream Western blotting. Excitation wavelengths include blue, green, violet and UV, while maximum emission wavelength is 610 nm. Fast protocol: Fix (1 hour) ► Stain (1 hour) ► Wash (30 min) ► Acidify (30 min) 1D and 2D gels stained with excellent sensitivity, large dynamic range, low background and no speckling! 16 Cat. no. Description Volume K-11072-B50 AdvanStain Scarlet Kit, sufficient for 20 minigels 5 mL K-11072-C25 AdvanStain Scarlet Kit, sufficient for 100 minigels 25 mL ► Image Sample Preparation ► Protein Separation ► ► Blotting AB Incubation ► Detection YY 3 Blotting Blotting is carried out to transfer proteins from the gel to a solid membrane. The blotting is performed by assembling filter papers, a membrane and a gel into a sandwich. An electrical field allows the proteins from the gel to wander onto the membrane. 3.1 Membranes Choose between nitrocellulose and PVDF membranes. We recommend pre-cut membranes for ease of use. Alternatively, cut your membranes from a roll for an economical solution. Use WesternBright transfer membranes for high performance and low background levels. WesternBright membrane characteristics: WesternBright PVDF-FL WesternBright PVDF-CL WesternBright NC Pore size 0.22 µm 0.22 µm 0.22 µm, 0.45 µm Thickness 135 µm 140 µm 150 µm Binding interaction Hydrophobic Hydrophobic Hydrophobic, electrostatic Typical protein binding capacity 300 µg/cm2 300 µg/cm2 200 µg/cm2 Fluorescence Total protein stain Total protein stain Chromogenic Chemiluminescence Chemiluminescence Direct staining Radiolabeling Colorimetric Electrophoretic transfer of proteins Electrophoretic transfer of proteins Electrophoretic transfer of proteins Protein dot and slot blotting Western transfer with fluorescent detection Western transfer with chemiluminescent detection Western transfer with chemiluminescent detection Colony/plaque lifts Pre-cut membranes Membrane rolls Pre-cut membranes Membrane rolls Pre-cut membranes Membrane rolls Detection methods Immobilization methods Recommended applications Available as Cat. no. Description Size L-08003-010 Pre-cut WesternBright NC 0.22 um, 8 x 10 cm 10 sheets L-08002-010 Pre-cut WesternBright NC 0.45 um, 8 x 10 cm 10 sheets L-08004-010 Pre-cut WesternBright PVDF-CL, 7 x 9 cm 10 sheets L-08001-010 Pre-cut WesternBright PVDF-FL, 7 x 9 cm 10 sheets L-08012-010 Pre-cut WesternBright PVDF-FL, 10 x 15 cm 10 sheets L-08011-010 Pre-cut WesternBright PVDF-CL, 10 x 15 cm 10 sheets L-08014-010 Pre-cut WesternBright PVDF-FL, 13 x 18 cm 10 sheets L-08013-010 Pre-cut WesternBright PVDF-CL, 13 x 18 cm 10 sheets L-08006-001 WesternBright NC membrane roll, 0.22 μm, 30 cm x 3 m 1 roll L-08007-001 WesternBright PVDF-FL membrane roll, 0.22 μm, 26 cm x 3.3 m 1 roll L-08008-001 WesternBright PVDF-CL membrane roll, 0.22 μm, 26 cm x 3.3 m 1 roll 17 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 3.2 Blotting Papers Standardized, pre-cut blotting papers save you time during sandwich assembly. We offer blotting papers with useful advantages: • • Pre-cut papers available in two sizes Uniform thickness ensures reproducible transfers without introducing artifacts or background noise Cat. no. Description Size L-07045-060 Blotting papers, 7 x 9 cm 60 sheets L-07056-060 Blotting papers, 8 x 10 cm 60 sheets 3.3 Transfer Buffer It is important to choose a high-quality transfer buffer to optimize the detection of low-abundance proteins, the transfer of high-molecular weight proteins and to save time. Optimize the transfer from gel to membrane with Advansta’s proprietary FLASHBlot Transfer Buffer: • • Ideal for low-abundance protein samples and post-translationally modified proteins Transfer proteins of all sizes in less than 20 minutes CEA (high MW) and GAPDH (low MW) Western blots. The proteins were transferred for 15 minutes using a FLASHBlot buffer and 60 minutes with a Towbin buffer, respectively. Transfers for both CEA and GAPDH were most efficient when using FLASHBlot. 18 Cat. no. Description Size R-03090-D25 FLASHBlot Transfer Buffer, 50x 250 mL R-03090-D50 FLASHBlot Transfer Buffer, 50x 500 mL Sample Preparation ► ► Protein Separation Blotting ► AB Incubation ► Detection YY 3.4 Protein Stain for Membranes Before proceeding with antibody incubation, it is advantageous to check the quality of the protein transfer. Advantages with AdvanStain Ponceau: • • • Stains your membrane in less than 5 min. Destain the membrane just as easily Following destaining, the membrane is ready for incubation with antibodies Cat. no. Description Size R-03021-D50 AdvanStain Ponceau 500 mL 19 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 4 Incubation with Antibodies Once the proteins have been transferred to a membrane, it needs to be blocked, incubated with a primary antibody, washed, incubated with a secondary antibody and then washed a final time. Instead of using both primary and secondary antibodies, direct detection can, in many cases, be more suitable. 4.1 Blocking Solution Avoid non-specific binding of antibodies by using an appropriate blocking solution. AdvanBlock-PF offers: • • • • Non-protein blocking solution No cross-reactivity with primary antibodies as seen with nonfat dry milk, BSA or casein Compatible with both fluorescent and chemiluminescent detection Optimized for use with WesternBright fluorescent secondary antibodies Cat. no. Description Size R-03023-D20 AdvanBlock-PF Blocking Solution, 5x 200 mL 4.2 Washing Buffer for Western Blots After each incubation step with antibodies, the blot needs to be washed with an appropriate washing buffer. Use AdvanWash washing solution whether you are performing fluorescent or chemiluminescent Western blotting. The solutions can be used with any Western blotting systems, but are optimized for use with WesternBright chemiluminescent HRP substrates, WesternBright MCF fluorescent Western blotting kit and WesternBright MCF-IR kits. • Reliable: Quality-controlled for reproducible results • Versatile: Compatible with chemiluminescent and fluorescent Western blots • Convenient: Save time using pre-made buffers Cat. no. Description Size R-03024-D50 AdvanWash Washing Solution, 10x 500 mL R-03100-D50 AdvanWash-IR Washing Solution, 10x 500 mL 4.3 Direct or Indirect Methods? Direct detection relies on a single primary antibody containing a detectable label, while indirect detection requires both a primary antibody as well as a secondary antibody. In indirect targeting, the secondary antibody contains the label. 20 Sample Preparation ► Protein Separation ► ► Blotting AB Incubation ► Detection YY Indirect Direct Advantages • Signal amplification – multiple secondary • Fewer steps antibodies bind to primary antibody • Less potential for non-specific binding • Versatile – same secondary antibody can be used on different primary antibody of same species Disadvantages • Risk of non-specific binding • More steps • Can be more costly • Labeling of antibody can affect the immunoreactivity 4.4 Primary Antibodies When performing Western blots, it is important to choose antibodies that recognize only the epitopes of interest. Follow the table below for advice on whether to choose monoclonal or polyclonal antibodies: Monoclonal antibodies Polyclonal antibodies Recognition of epitope • Recognize a single epitope • Recognize multiple epitopes within same antigen Sensitivity • Less sensitive • More sensitive – good for low abundance antigens Cross-reactivity • Less likely • Potential for recognizing other antigens with the epitope • More likely • Higher background than monoclonal antibodies • Can cross-react with multiple animal species Variability • Very stable batches from same hybridoma • Variability-prone between batches Tolerance* • Less tolerant • More tolerant *Tolerance against differing conditions, such as denaturing conditions, modifications or presence of polymorphism 4.5 Secondary Antibodies For indirect detection, the blot is treated with secondary antibodies after primary antibody incubation. The primary antibody species determines the choice of secondary antibodies. For chemiluminescence, secondary antibodies are labeled with HRP, while for fluorescence they are labeled with a fluorophore. We offer secondary antibodies from a variety of species with different conjugates. HRP-conjugated secondary antibodies for chemiluminescent detection: Cat. no. Description Size R-05071-500 Goat anti-mouse HRP-conjugated secondary antibody 500 µL R-05072-500 Goat anti-rabbit HRP-conjugated secondary antibody 500 µL R-05073-500 Goat anti-human HRP-conjugated secondary antibody 500 µL R-05074-500 Goat anti-chicken HRP-conjugated secondary antibody 500 µL R-05075-500 Goat anti-rat HRP-conjugated secondary antibody 500 µL R-05076-500 Goat anti-guinea pig HRP-conjugated secondary antibody 500 µL 21 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY Fluorescent secondary antibodies, excitable with visible and near infrared light: Cat. no Description, visible light Size R-05051-050 Goat anti-rabbit IgG APC 50 µL R-05051-250 Goat anti-rabbit IgG APC 250 µL R-05052-050 Goat anti-mouse IgG RPE 50 µL R-05052-250 Goat anti-mouse IgG RPE 250 µL Cat. no. Description, infrared light Size R-05054-250 Goat anti-rabbit IgG IR700 250 µL R-05055-250 Goat anti-mouse IgG IR700 250 µL R-05056-250 Goat anti-human IgG IR700 250 µL R-05057-250 Goat anti-chicken IgY IR700 250 µL R-05058-250 Goat anti-rat IgG IR700 250 µL R-05059-250 Goat anti-guinea pig IgG IR700 250 µL R-05060-250 Goat anti-rabbit IgG IR800 250 µL R-05061-250 Goat anti-mouse IgG IR800 250 µL R-05062-250 Goat anti-human IgG IR800 250 µL R-05063-250 Goat anti-chicken IgY IR800 250 µL R-05064-250 Goat anti-rat IgG IR800 250 µL R-05065-250 Goat anti-guinea pig IgG IR800 250 µL Secondary antibodies for a large selection of species: From Santa Cruz Biotechnology we offer a wide variety of conventional secondary antibodies for a large selection of species, raised in either rabbits, goats, donkeys, bovines, mice or chickens. • Anti-Goat • Anti-Mouse • Anti-Rabbit • Anti-Rat • Anti-Bovine • Anti-Cat • Anti-Chicken • Anti-Dog • Anti-Guinea Pig • Anti-Armenian Hamster • Anti-Horse • Anti-Human • Anti-Monkey • Anti-Swine • Anti-Turkey • Anti-Sheep • Anti-Syrian Hamster • Anti-Donkey Available conjugated to either an enzyme, biotin or fluorophore. Cruz Marker™-compatible Western blotting secondary antibodies and Cruz Marker™ Molecular Weight Standards. Cruz Markers™ are provided for use as internal molecular weight standards for chemiluminescence Western blotting applications, and they are ideal for approximating the molecular weight of target proteins while allowing researchers to visualize the final molecular weight ladder of six bands clearly and consistently on the final Western blot film. Cruz Marker™ compatible secondary antibodies recognize an epitope common to each of the Cruz Marker™ molecular weights. Pre-adsorbed secondary antibodies are recommended for Western blotting of immunoglobulin-rich tissues and cells. 22 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY Cruz Marker™ Molecular Weight Standards* Description Cat. no Size Cruz Marker™ Molecular Weight Standards sc-2035 200 µL *Must be used in connection with Cruz Marker™-compatible Western blotting secondary antibodies. Cruz Marker™-compatible Western blotting secondary antibodies* Description HRP conjugate AP conjugate HRP conjugate Pre-adsorbed AP conjugate Pre-adsorbed Bovine anti-goat IgG sc-2378 sc-2381 sc-2384 sc-2387 Donkey anti-goat IgG sc-2033 sc-2037 sc-2304 sc-2310 Bovine anti-mouse IgG sc-2380 sc-2383 sc-2386 sc-2389 Donkey anti-mouse IgG sc-2318 sc-2320 sc-2306 sc-2312 Goat anti-mouse IgG sc-2031 sc-2047 sc-2302 sc-2308 Bovine anti-rabbit IgG sc-2379 sc-2382 sc-2385 sc-2388 Donkey anti-rabbit IgG sc-2317 sc-2319 sc-2305 sc-2311 Goat anti-rabbit IgG sc-2030 sc-2034 sc-2301 sc-2307 Goat anti-rat IgG sc-2032 sc-2036 sc-2303 sc-2309 Rabbit anti-mouse IgG sc-358917 sc-358923 *Are supplied at 200 µg in 0.5 mL, to be used in Western blotting at a dilution of 1:500–1:5000. 4.6 Streptavidin Conjugates The strong interaction between streptavidin and biotin can be utilized for Western blotting. Using streptavidin conjugates enables fluorescent detection of biotinylated proteins. Fluorescent streptavidin conjugates: Cat. no. Description Size R-05011-050 AdvanFluor APC-Streptavidin conjugate 50 µL R-05012-050 AdvanFluor BPE-Streptavidin conjugate 50 µL R-05011-200 AdvanFluor APC-Streptavidin conjugate 200 µL R-05012-200 AdvanFluor BPE-Streptavidin conjugate 200 µL We offer over 70,000 monoclonal and polyclonal primary and secondary Western blot antibodies from our suppliers. Antibodies for cancer research, cardiovascular research, drug discovery, immunology, metabolism, neuroscience research, etc. 23 Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 5 Detection The detection method is dependent on the label of the antibodies. Using a fluorescence dye enables detectable excitation by light. For chemiluminescent detection, HRP-labeled antibodies are treated with substrate, which produces the detectable signal. 5.1 HRP Substrates Most Western blot users have special needs for HRP substrates, including high sensitivity, long-lasting signal, fast detection and higher dynamic range. Choose the right product for the chemiluminescent detection of your target proteins. 24 Substrate Use Sensitivity Signal duration WesternBright ECL Sensitive, economical choice for routine Western blotting Low picograms 6-8 hours WesternBright Quantum Extended dynamic range for detecting low and high abundance proteins within the same blots Mid femtograms 10-24 hours WesternBright Sirius Low abundance protein detection Low femtograms 6 hours Cat. no. Description Size K-12045-D20 WesternBright ECL 200 mL K-12045-D50 WesternBright ECL 500 mL K-12049-D50 WesternBright ECL spray 500 mL K-12042-D10 WesternBright Quantum 100 mL K-12042-D20 WesternBright Quantum 200 mL K-12043-D10 WesternBright Sirius 100 mL K-12043-D20 WesternBright Sirius 200 mL Sample Preparation ► Protein Separation ► Blotting ► AB Incubation ► Detection YY 5.2 How to Choose the Right Chemistry for Your Western Blot The best chemistry for your Western blot depends on the type of information you are looking for. 5.2.1 Chemiluminescence Chemiluminescent Westerns are relatively easy to do and can be extremely sensitive; substrates are available to detect proteins in the femtogram range. “Chemi” is a convenient chemistry if proteins of interest differ significantly in molecular weight and are easily resolved by gel electrophoresis. Advantages: Disadvantages: • Sensitive • Easy, familiar chemistry • Compatible with film or digital imaging • Semi-Quantitative • Signal dependent on enzyme kinetics • Single protein only, loading controls require stripping and re-probing Use Chemiluminescence to: • • • • • Detect a single protein Assay for presence/absence of a protein Measure antibody responses Follow protein purification Detect low abundance proteins Chemiluminescence. One signal. One protein 5.2.2 Fluorescence Fluorescent Western blotting uses secondary antibodies conjugated to fluorescent dyes. The amount of light emitted from excited fluorophores is proportional to the amount of target protein on the membrane. This allows for true quantitative analysis. Using fluorophores with different emission spectra, one can detect multiple proteins on a blot simultaneously (multiplexing). Loading controls can be measured on the same blot as your protein of interest, along with post-translational modifications without stripping and re-probing. Fluorescent Westerns use either Infrared or Visible fluorophores. Advantages: Disadvantages: • Multiplex capability • Increased quantitative accuracy • Fluorescent label stability allows blots to be stored and re-imaged later • Can be less sensitive than chemiluminescence • Membrane auto-fluorescence can increase background Advantages of Visible Fluorescent Detection If you need to study three proteins at once, visible fluorescence is the obvious choice. Visible fluorescence also offers flexibility. There are many choices of fluorophores available for visible detection. If you are already using (spectrally separated) visible fluorophores for other experiments, adapting these for use in fluorescent Western blots is easy. 25 ► Sample Preparation Protein Separation ► Blotting ► AB Incubation ► Detection YY Use Fluorescence to: • • • • • Detect multiple proteins simultaneously Study post-translational modifications Have same-blot loading control Have in-lane normalization Perform quantitative Westerns Multiplex Fluorescence. Multiple Proteins The Azure c Series Imaging Systems The Azure Biosystems cSeries offers 6 unique gel imaging systems. Select the one that fits your applications now, and upgrade later when your needs change. • A total solution for Western Blot Imaging and Gel Documentation: The cSeries is a multichannel, multimodal imager, with IR, visible light, and UV excitation channels. • Compact, integrated design: A unique design and integrated tablet computer gives this system a small footprint, saving you room for other tools on the bench top. • Fully upgradable: All models can be easily upgraded at a later time to accommodate additional applications. • 8.3MP CCD camera for sensitive detection of chemiluminescence: 5 levels of binning and a Chemi Blot Shelf to bring the blot closer to the detector, if needed. • 16 bit camera: c-series has a wide dynamic range, preventing saturation and keeping results in linear range. • Deep Peltier cooling of the camera: Absolute and Regulated cooling improves image quality by reducing the noise in the image. • AzureSpot Analysis Software: Analysis tool for 2D densitometry, automatic lane and band detection, background correction, normalization, molecular weight calculation, and annotation. c600 √ c500 √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ √ c200 √ √ √ c150 √ √ √ c400 c300 26 √ √ 6 Troubleshooting 6.1 Unusual/Unexpected Bands Problem Lower MW than expected Slightly higher MW than expected Significantly higher MW than expected Cause Solution • Digestion/cleavage of target • Use fresh sample and protease inhibitors • Splice variant exist or other proteins bear similar epitope • Consult literature and use appropriate controls • Try another antibody • Potential for glycosylated protein or small amino acid modifications • Use enzymes to remove the modification • Check amino acid sequence and literature • Dimers/multimers/protein-protein interactions occur due to inefficient reduction and denaturation • Use fresh DTT or β-mercaptoethanol for samples + reheat before repeating experiment • Prepare new samples with fresh loading buffer • Changed protein expression during over-passaged cell line • Use earlier passages, include positive control • Excessively high concentration of primary antibody or occurrence of cross-reactivity with similar epitopes on other proteins • • • • • Non-specific binding of secondary antibody • Optimize secondary antibody concentration • Use affinity-purified secondary antibody • Check non-specific binding by repeating immunodetection with secondary antibody alone • Voltage too high during gel run • Run gel at lower voltage Multiple bands Blurry/diffuse bands Smiling bands (not flat) White bands Streaking in lanes Lateral spreading of bands Band distortion Use affinity-purified primary antibody Optimize primary antibody concentration Try another Check antibody specificity with blocking peptide • Slow migration • Increase voltage, ensure proper buffer preparation • Incorrect heating of sample • Ensure heating to 90 °C for 2 min prior to loading • Old SDS in sample buffer • Use fresh SDS for sample buffer • Incorrect buffer composition • Use fresh buffer • Bubbles trapped between gel and membrane during transfer • Carefully remove air bubbles • Overheated gel due to fast running conditions • Optimize electrophoresis conditions • Run gel at 4 °C • Too much protein loaded • Decrease amount of sample • Repeat with dilution series of sample • Antibody concentration too high • Optimize antibody concentrations • High salt concentration in sample • Decrease salt concentration in sample buffer • Sample concentration too high/insufficient SDS • Increase dilution/add more SDS • Diffusion of samples from well to well • Minimize loading time • Incomplete polymerization of gel around wells • Increase TEMED/AP • High pressure applied to gel during pouring • Loosen screws on the gel assembly apparatus • Particulate matter in gel • Filter and mix gel reagents prior to gel preparation • Excessive/uneven heating of gel • Decrease running voltage/apply cooling during run • Bubbles in gel due to dirty plates • Wear gloves during plate handling • Clean plates thoroughly with ethanol and deionized water • Bubbles in gel from air introduced from pouring device • Do not expel entire volume of gel mix • Bubbles under comb • Insert comb at an angle and reposition before gel solidification 27 6.2 No Bands, Faint Bands or Weak Signal Problem Sample preparation Inadequate transfer Inefficient binding of primary antibody Inefficient binding of secondary antibody Inactive conjugate/substrate Detection methods Cause Solution • Inefficient extraction method • Use other extraction methods • Include positive control • Low expression levels of protein • Load more protein, concentrate extracted proteins, pool multiple samples • Degraded protein during degradation • Use fresh protease inhibitor • Wrong transfer buffer composition, too much methanol in buffer • Check protocol, decrease methanol concentration • Large protein requires more time and/or current • Run for longer time and/or with higher voltage • No contact between gel and membrane • Check fiber pad thickness, replace if too thin • Protein has transferred through the membrane • Use two membranes • Old/weak antibody, low affinity of antibody for protein • Increase primary antibody concentration, purchase fresh antibody, reduce number of wash steps • Weak cross-reactivity with species of interest • Try other primary antibody source • Antibody removed with washing steps • Decrease number of washes, decrease salt concentration in wash buffer, reduce washing time • Antigen masked by blocking agent • Use alternative blocker • Try lower concentration of blocker • Inappropriate secondary antibody • Use antibody directed against primary antibody species • Insufficient antibody concentration/antibody too old • Purchase new antibody • Increase dilution • Old/unstable reagents • Check conjugate and substrate for signal • Purchase new reagent • HRP inactivation by sodium azide • Avoid sodium azide • Old detection reagent/improper storage of detecting reagent • Purchase new reagent • Exposure time too short • Test different exposure times 6.3 Electrophoretic Problems Problem Solution • Insufficient TEMEP/AP • Increase amount of TEMEP/AP Slow/No gel polymerization • Old AP solution • Prepare fresh AP and store only for a few days at 4 °C • Polymerization inhibition due to oxygen • Layer gel with isopropanol prior to pouring stacker Too fast gel polymerization • Excessive amount of TEMED/AP • Decrease amount of TEMED/AP • Concentration of running buffer too high • Check protocol/dilute buffer • Insufficient current • Increase voltage • Buffer excessively diluted • Check protocol/replace buffer Smiling dye front • Migration too fast • Decrease voltage • Heat generation • Run with cooling Slanted dye front • Bubble between glass plates at the bottom of the gel • Hold gel at an angle, place corner into lower buffer chamber and slowly move to normal position Very long run time Running time unusually short 28 Cause 6.4 High Background Problem Membrane blocking is insufficient Inappropriate wash conditions Reagant contamination Membrane choice Overexposed image Cause Solution • Biotin in milk incompatible with streptavidin system/milk contains antigen of interest • Try another blocking agent • Milk solution excessively diluted • Increase milk solution • Blocking time too short • Increase incubation time • Some detergents are not as effective at cold temperatures • Incubate for 1 hour at room temperature instead of overnight at 4 °C • Insufficient detergent concentration • Use stronger detergents/increase detergent concentration • Insufficient washing • Increase duration of washing steps, use larger volume of washing buffer, increase number of washing steps • Try other incubation temperatures • Bacterial/fungal growth in buffers • Check turbidity in buffers • Prepare new buffers • PVDF membranes may have higher background than nitrocellulose • Try nitrocellulose membranes • High autofluorescence in membrane • Use low autofluorescence PVDF membranes for fluorescent Western blots • Membrane has dried out • Ensure hydration during all steps by using sufficient volumes and agitation • Exposure time too long • Reduce exposure time, increase antibody dilutions • Load less sample Non-specific bin- • Antibody concentration too high/antibody not affinity purified ding of primary or secondary • Too much protein on gel antibodies • Decrease antibody concentration/try monoclonal or affinity-purified antibody/optimize incubation times • Load less protein 29 Contact Denmark blå tynd streg hvid alm. streg blå alm. streg pink tynd streg +45 8745 9010 www.ahdiagnostics.dk [email protected] Sweden blå tynd streg hvid alm. streg blå alm. streg pink tynd streg +46 08 680 08 45 www.ahdiagnostics.se [email protected] Norway blå tynd streg hvid alm. streg blå alm. streg pink tynd streg +47 2323 3260 www.ahdiagnostics.no [email protected] Finland blå tynd streg hvid alm. streg blå alm. streg pink tynd streg +358 8 010 325 3000 www.ahdiagnostics.fi [email protected] 30 31 AH diagnostics as • Gammel Viborgvej 11A • 8381 Tilst • Tel.: +45 8745 9010 • [email protected] • www.ahdiagnostics.dk AH diagnostics AB • Vallgatan 9 • SE-170 67 Solna • Tel.: +46 (0)8 680 0845 • [email protected] • www.ahdiagnostics.se AH diagnostics Oy • Viikinkaari 4 • FI-00790 Helsinki • Tel.: +358 (0)10 325 3000 • [email protected] • www.ahdiagnostics.fi AH diagnostics as • Fjellgata 1 • NO-0566 Oslo • Tel.: +47 2323 3260 • [email protected] • www.ahdiagnostics.no D S N F

Focus Western Blot

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