Step 4. Step 4. Choose MW markers. Choose MW markers. Gel Electrophoresis of Proteins
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1 Gel Electrophoresis of Proteins Choose MW markers 16 Step 4 Gel Electrophoresis of Proteins A Practical Approach, Third Edition An overview of basic techniques to benefit any life scientist using electrophoretic methods. Includes emerging techniques such as capillary gel electrophoresis, sequence analysis of gel-resolved proteins, fluorophorelabeled saccharide electrophoresis, and use of gel electrophoresis for analysis of protein:protein interactions. Step 4 Choose MW markers Molecular Weight Markers To assess relative molecular weight of a protein on a gel, protein molecular weight markers are run in outer lanes of for comparison. A standard curve can be constructed from distances migrated by each marker protein. The distance migrated by unknown protein is n plotted and molecular weight is interpolated from standard curve. Pierce offers a variety of molecular weight (MW) markers for use with one- and twodimensional gels and for various detection methods. Table 3 summarizes different features of each marker mix. These protein marker mixes are available unstained for in-gel staining with any protein stain or as prestained markers that can be visualized in a gel or when transferred to a membrane. The most convenient feature of Ranger Molecular Weight Markers is unique room temperature-stable, single-dose tube packaging. There is no need to aliquot and freeze marker formulation because it is already dispensed into a 48-microtube tray. Simply puncture protective foil layer and add running buffer to rehydrate proteins. Then transfer 2-10 µl of mix to a lane on. The 2-D Protein Molecular Weight Marker Mix (Product # 26659) provides a unique protein formulation with a wide pi range ( ) for analysis in first dimension and a wide MW range (17 K-80 K) for second dimension. The Chemiluminescent BlueRanger Protein Molecular Weight Marker Mix (Product # 26651) has been created for chemiluminescent detection on Western blots. This MW marker shows up in-gel and on a membrane because it is prestained. It also can be detected on film using a chemiluminescent substrate for HRP because it is peroxidase-labeled. Hames, B.D., Ed., Published by Oxford University Press, 1998, 352 pages, Soft-cover Product #: 20044
2 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers Table 3. Pierce protein molecular weight markers products for electrophoresis.* Run Stain Poststaining BlueRanger TriChromRanger ColorMeRanger Chemiluminescent DyLight MW Markers Markers Markers BlueRanger Markers 2-D MW Marker Mix Marker Mix Protein (Product # 26681) (Product # 26691) (Product # 26671) (Product # 26651) (Product # 26659) (Product # 26665) Myosin 210 K 210 K 200 K 220 K 200 K Phosphorylase B 120 K 110 K 97.4 K 104 K 97 K ApoTransferrin 80 K Bovine Serum 84 K 80 K 66K 76 K 66 K Albumin (BSA) Glutamic 56 K Dehydrogenase Ovalbumin 60 K 47 K 43 K 45 K Protein A 42 K Actin 43 K Carbonic Anhydrase 39.2 K 32 K 29 K 33 K 29 K Protein L 36 K Myokinase 22.5 K Peanut Agglutinin 28 K Soybean Trypsin 28 K 25 K 20 K 26 K 20 K 20 K Inhibitor Myoglobin 17 K Lysozyme 18.3 K 16.5 K 14.4 K 18 K 14 K Aprotinin 6 K 6 K Insulin B Chain 3.5 K Staining Feature Prestained (1 color) Prestained (3 colors) Unstained Peroxidase labeled Unstained Prestained and prestained (2 fluors) (1 color) Package 48 microtubes 48 microtubes 48 microtubes 48 microtubes 500 µl (~250 gels) 250 µl (50 gels) *Actual molecular weights are lot-specific because proteins are prestained. Lot-specific information is included in each package. New! Step 4 Choose MW markers 17 BlueRanger Prestained Molecular Weight Markers 15,16 A fresh marker every time, not just first time. A totally new idea in how molecular weight markers are packaged! Innovative single-dose package Room temperature stable Excellent performance on wide range of gel compositions Efficient membrane transfer Single-dose packaging in a novel microtube plate format eliminates opportunities for contamination due to multiple marker withdrawals from same vial Unique stabilized prestained markers can be stored at room temperature Compatible with a broad range of SDS-PAGE gel compositions and downstream applications Prestained proteins transfer well to both nitrocellulose and PVDF membrane Can be used with GelCode Blue, SilverSNAP and E-Zinc Stains Formulated to yield prestained protein bands of equal intensity Here s how it works: BlueRanger Prestained Markers are an individually dried and stabilized formulation of seven proteins spanning range from 18.3 K to 210 K. The plate is covered with a foil that can be easily punctured with a pipette tip. Simply puncture foil covering a single well containing dried marker mix with a pipette tip and add 10 µl deionized water. The marker proteins are reconstituted instantly and ready for loading onto a gel lane. The proteins listed, covering a broad molecular weight range, have been prestained and purified to give single bands on 4-20% SDS-PAGE gels. Each protein has been proportioned into mix to yield uniform band intensity. Tel: or
3 Step 4 Choose MW markers Gel Electrophoresis of Proteins BlueRanger Prestained Molecular Weight Markers, continued Markers are ready when you are and room temperature-stable. 1. Open resealable plastic pouch and remove BlueRanger Prestained Protein Molecular Weight Marker Mix. BlueRanger Prestained Marker Mix is packaged with a desiccant in a moisture-resistant, resealable pouch. 2. Load 10 µl of DI water into a pipette tip, puncture foil over a single tube and dissolve BlueRanger Prestained Markers. 3. Dispense 5-10 µl of marker into a sample well of to be run. Each tube can be used to prepare one or two lanes of a gel. BlueRanger Prestained Marker Protein Molecular Weights Myosin Phosphorylase B Serum Albumin Ovalbumin Carbonic Anhydrase Trypsin Inhibitor Lysozyme 210 K 120 K 84 K 60 K 39.2 K 28 K 18.3 K Return BlueRanger Prestained Marker Mix to its pouch and reseal. The markers are stable at room temperature and can be kept right on your bench-top ready for your next SDS-PAGE gel. References 15. Foubert, T.R., et al. (2001). J. Biol. Chem. 276, Prozialeck, W.C., et al. (2002). Infect. Immun. 70, BlueRanger Prestained Protein 1 x 48 Molecular Weight Marker Mix microtube Sufficient material for loading plate gel lanes BlueRanger Prestained Protein 5 x 48 Molecular Weight Marker Mix microtube Sufficient material for loading plates gel lanes. TriChromRanger Prestained Molecular Weight Markers 17,18 Fresh marker every time, with reference bands too. BlueRanger Marker Myosin (210 K) Phosphorylase B BSA Ovalbumin TriChromRanger Marker Innovative single-dose packaging allows you to dissolve only marker you need exactly when you want it The single-dose packaging eliminates possibility of contamination due to multiple withdrawals Room-temperature storage eliminates need to expose protein markers to detrimental freeze-thaw cycles References 17. Myers, C.R. and Myers, J.M. (2002). Appl. Envir. Microbiol. 68, Cui, L., et al. (2002). Am. J. Physiol. Cell Physiol. 283, C623-C630. Carbonic Anhydrase Trypsin Inhibitor Lysozyme (16.5 K) Each tube of TriChromRanger Marker consists of a stabilized and lyophilyzed formulation of seven proteins, ranging from 16.5 K to 210 K. Each protein in mixture is proportioned to yield uniform band intensities. Two specially modified bands (one red, one violet) serve as references for order of marker proteins TriChromRanger Prestained Protein 1 x 48 Molecular Weight Marker Mix microtube Sufficient material for loading gel lanes. plate For more product information, or to download a product instruction booklet, visit
4 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers Chemiluminescent BlueRanger Molecular Weight Markers Run Stain Poststaining New protein molecular weight standard looks and acts like a typical pre-stained marker for SDS-PAGE and can also light up after transfer or in-gel. Colorimetric and chemiluminescent two detection options are available: on-membrane or in-gel Visual detection in-gel already prestained; does not require staining to detect in-gel Universal compatibility with HRP conjugates self-contained peroxidase activity, does not require an HRP-antibody conjugate for chemiluminescence and no variability due to host animal or antibody class Compatible with streptavidin-hrp conjugates Room temperature stable Convenient packaging single dose in 48-well microtube plate The Chemiluminescent BlueRanger Marker consists of seven proteins spanning molecular weight range from 18 K to 220 K. Each marker component is prestained covalently with a blue dye followed by chemical modification imparting peroxidase capability. Unlike any or chemiluminescent detection-compatible marker for Western blot applications, Chemiluminescent BlueRanger Marker does not depend on binding of an HRP-antibody conjugate to it to yield a chemiluminescent signal. Component Proteins Myosin Heavy Chain Phosphorylase B BSA Ovalbumin Carbonic Anhydrase Trypsin Inhibitor Lysozyme A. B. Colorimetric and Chemiluminescent Detection on a Western Blot MW of Chemiluminescent BlueRanger Proteins* 220 K 104 K 76 K 45 K 33 K 26 K 18 K Colorimetric and Chemiluminescent Detection In-Gel using UnBlot Technology *These are representative molecular weight values. The covalently bound dye and enzyme alter apparent molecular weight (MW) of component proteins relative to ir unstained counterparts. Lot-specific MW values are provided with each package. Figure 5. A. Western blot detection. Lanes 1-4 show marker run on a 4-20% Tris-Glycine SDS-polyacrylamide gel and transferred to nitrocellulose. Lanes 1 and 3 were loaded with 2 µl of marker. Lanes 2 and 4 were loaded with 5 µl of marker. Lanes 1 and 2 marker colorimetrically after transfer to membrane. Lanes 3 and 4 were treated with SuperSignal West Pico Chemiluminescent Substrate (Product # 34080) and exposed to X-ray film for 1 minute. B. In-gel detection (UnBlot Technology ). Lanes 1 and 2 were each loaded with 10 µl of marker before electrophoresis on a 4-20% Tris-Glycine gel. Lane 1 shows marker bands colorimetrically in-gel. Lane 2 shows marker proteins detected in-gel using UnBlot Technology with UnBlot Chemiluminescent Substrate (Product # 33550) and exposure of to X-ray film for one minute. Chemiluminescent marker optimized for Western blot and Pierce UnBlot Applications Figure 6. Pierce Chemiluminescent BlueRanger Markers (5 µl and 2 µl loading) (Lanes 1 and 2) and two 6xHis proteins (~10 ng) (Lanes 3 and 4) were separated by electrophoresis using a 4-20% Tris-Glycine gradient gel. The proteins were transferred to nitrocellulose and detected for 6xHis tag using SuperSignal West HisProbe Kit (Product # 15168). The blot was exposed to X-ray film for 1 minute to capture chemiluminescent signal. (The blot was scanned to document color.) Chemiluminescent BlueRanger 1 x 48 Prestained Peroxidase-Labeled microtube Protein Molecular Weight Marker Mix plate CAUTION: Chemiluminescent BlueRanger Markers are prelabeled with a peroxidase enzyme. This means y must be handled more gently than traditional prestained markers. These markers can be overheated to point of inactivation during transfer from to membrane. They can also be inactivated by or conditions that are detrimental to peroxides such as too much EDTA/EGTA, azide or acidic membrane stains such as Ponceau S. In most systems, inactivation is unlikely to occur, but if it does occur in your system, please return remaining product for a full refund. Step 4 Choose MW markers 19 Tel: or
5 Step 4 Choose MW markers 20 Gel Electrophoresis of Proteins 2-D Protein Molecular Weight Markers A 2-D gel marker mix that offers a broad range of pi and MW. The new ready-to-use 2-D gel marker mix from Pierce is designed specifically to aid proteome analyst. This 2-D gel marker mix contains a complement of seven reduced and denatured proteins. When performing protein 2-D separation and analysis, each protein in mix provides important features for assessing system performance or estimating pi and molecular weight values. It s also convenient this marker mix doesn t require you to sign a notarized document just to use it. A unique complement of proteins Each protein in this marker mix was carefully selected to give a useful range of molecular weight and pi coverage. Proteins were selected that give a variety of features from tight single spots to characteristic charge trains to aid analyst in gel orientation. Molecular weights range from 17 K to 80 K, with pi values ranging from 4.5 to 8.7. Component Proteins in new Pierce 2-D Protein Molecular Weight Marker Mix 2-D Marker Protein MW pi Value Apo-Transferrin (human plasma) 80 K 6.2 Glutamic Dehydrogenase (bovine liver) 56 K 6.5, 6.7, 6.9 Actin (bovine muscle) 43 K 5.2 Carbonic Anhydrase (bovine erythrocytes) 29 K 6.3 Myokinase (chicken muscle) 22.5 K 8.7 Trypsin Inhibitor (soybean) 20 K 4.5 Myoglobin (equine skeletal muscle) 17 K 7.0, 7.4 F C A D B A. Human Apo-Transferrin E. Chicken Myokinase B. Bovine Glutamic Dehydrogenase F. Soybean Trypsin Inhibitor C. Bovine Muscle Actin G. Equine Myoglobin D. Bovine Carbonic Anhydrase Figure 7. 2-D markers shown stained with silver (left) and coomassie blue dye (right). 500 µl of Pierce 2-D Marker Mix is sufficient for following number of applications, depending on gel size and staining method Stain Marker # of Gels/ Gel Size Method Volume Vial Mini-Gels Coomassie Blue Dye 2.5 µl 200 Mini-Gels Silver µl 500-1,000 Large Format Gels Coomassie Blue Dye µl Large Format Gels Silver µl The 2-D Protein Molecular Weight Marker Mix is supplied frozen. For optimal long-term stability, aliquot into sample vials upon receipt and refreeze. G E F C A D B G E D Protein Molecular Weight 500 µl Marker Mix For more product information, or to download a product instruction booklet, visit
6 Electrophoresis & Staining Flowchart 1. sample Buffers Choose MW markers DyLight Fluorescent Protein Molecular Weight Markers One- or two-color fluorescent detection with one protein molecular weight marker. DyLight Fluorescent Protein Molecular Weight Markers are optimized for direct visualization of marker proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Each protein in mixture is double-labeled with DyLight 547 and DyLight 647 Fluorescent Dyes to provide flexible one- or two-color detection with common imaging systems (Figure 8). The markers are compatible with Western blotting (Figure 9) and can be detected by virtually any in-gel staining method (Figure 10). The DyLight Fluorescent Protein Molecular Weight Markers consists of nine proteins with molecular weights in range of 6 K to 200 K. Fluorescent and Colorimetric Detection of DyLight Fluorescent Protein Molecular Weight Markers Myosin (200 K) Phosphorylase B (97 K) BSA (66 K) Protein A (42 K) Protein L (36 K) Peanut Agglutinin (28 K) Trypsin Inhibitor (20 K) Lysozyme (14 K) Aprotinin (6 K) Figure 8 A B Figure 9 A B Figure 10 A B Run Stain Poststaining Easily multiplexed two excitation and emission maxima enable one- or two-color fluorescent detection Easy to use and convenient eliminate need for awkward marking or overlay procedures Fluorescent and colorimetric two detection options available: in-gel or on-membrane Instrument-compatible DyLight Dye spectra are compatible with common imaging systems Photostable allows long exposure times for maximum sensitivity Table 4. Spectral characteristics of DyLight Fluorescent Protein Molecular Weight Markers. Extinction Excitation (nm) Emission (nm) Coefficient (min) DyLight 547 Dye ,000 M -1 cm -1 DyLight 647 Dye ,000 M -1 cm DyLight Fluorescent Protein 250 µl Molecular Weight Markers Sufficient material for loading 50 gel lanes. Step 4 Choose MW markers 21 Figure 8. Direct in-gel fluorescent detection. Marker proteins (5 µl) were separated in 4-20% Tris-glycine gels. The gels were imaged with Kodak Image Station 2000MM using a five-minute exposure at f2.8 with A. 535/600 nm excitation/emission filter set or B. 625/700 nm excitation/ emission filter set. Figure 9. Fluorescent Western blot detection. Marker proteins (5 µl) were separated in 4-20% Tris-glycine gels and transferred to A. nitrocellulose or B. PVDF membrane. Blots were imaged with Typhoon 9410 at 500V PMT using A. Cy 3 Dye or B. Cy 5 Dye laser settings. Note: Proteins in marker mix produce uniform fluorescent intensities in SDS-PAGE applications; however, variations in protein-transfer efficiency affect fluorescent intensity. For example, high molecular weight proteins, such as myosin (200 K), typically transfer less efficiently than low molecular weight proteins. Figure 10. Colorimetric in-gel detection. Marker proteins (10 µl) were separated in 4-20% Tris-glycine gels and stained with A. Imperial Protein Stain or B. silver stain. Tel: or
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