Instruction Manual And Application Guide - Bio-Rad
Mini-PROTEAN Precast GelsInstruction Manual and Application Guide
Bio-Rad Technical SupportFor help and technical advice, please contact the Bio-Rad Technical Support department. In the United States, the TechnicalSupport department is open Monday–Friday, 5:00 am–5:00 pm, Pacific Time.Phone: 1-800-424-6723Fax: 1-510-741-5802Email: LSG TechServ US@bio-rad.com (for U.S. and international customers)Online technical support and worldwide contact information are available at www.consult.bio-rad.com.Legal NoticesNo part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, includingphotocopy, recording, or any information storage or retrieval system, without permission in writing from Bio-Rad Laboratories.Bio-Rad reserves the right to modify its products and services at any time. This user guide is subject to change without notice.Although prepared to ensure accuracy, Bio-Rad assumes no liability for errors, or for any damages resulting from the applicationor use of this information.Coomassie is a trademark of BASF Aktiengesellschaft. Ficoll is a trademark of GE Healthcare Group companies. StrepTactinis a trademark of Institut für Bionalytik GmbH. StrepTactin is covered by German patent application P 19641876.3. Bio-RadLaboratories, Inc. is licensed by Institut für Bioanalytik GmbH to sell these products for research use only. SYBR is a trademarkof Invitrogen Corporation. SYPRO is a trademark of Molecular Probes, Inc. Bio-Rad is licensed to sell SYPRO products forresearch use only, under U.S. Patent 5,616,502. Tween is a trademark of ICI Americas, Inc.Copyright 2011 by Bio-Rad Laboratories. All rights reserved.
ContentsChapter 1: Mini-PROTEAN Precast Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11.2 Gel Formulations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.3 Comb Configurations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.4 Specifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21.5 Storage Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31.6 Important Notes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3Chapter 2: Setup and Basic Operation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.1 Workflow Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2 Required Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.3 Setting Up and Running Mini-PROTEAN Gels in the Mini-PROTEAN Tetra Cell . . . . . . . . . . . . . . . 52.4 Removing the Gel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7Chapter 3: SDS-PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83.2 Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free Gels . . . . . . . . . . . . . . . . . . . . . . . . 83.3 SDS-PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 103.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10Chapter 4: Native PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.2 Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.3 Native PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13Chapter 5: Stain-Free System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145.2 Stain-Free Workflow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155.3 Electrophoresis with Mini-PROTEAN TGX Stain-Free Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 155.4 Stain-Free Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15
Chapter 6: Peptide Analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.2 Mini-PROTEAN Tris-Tricine Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.3 Peptide Analysis Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17Chapter 7: Nondenaturing Nucleic Acid PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.2 Mini-PROTEAN TBE Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.3 Nondenaturing Nucleic Acid PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Chapter 8: Denaturing Nucleic Acid PAGE . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208.2 Mini-PROTEAN TBE-Urea Gels . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208.2.1 Gel Composition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208.2.2 Gel Selection Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208.3 Denaturing Nucleic Acid PAGE Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218.4 Sample Preparation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 218.5 Running Conditions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21Chapter 9: 2-D Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229.2 Equilibration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229.3 Agarose Overlay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 229.4 Second-Dimension Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22
Chapter 10: Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2310.1 SDS-PAGE and Native PAGE Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2310.2 Peptide Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2410.3 TBE Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2410.4 TBE-Urea Gel Staining . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 24Chapter 11: Blotting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2511.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2511.2 Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2511.2.1 Transfer Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2511.2.2 Wet Transfer Using the Mini Trans-Blot Module . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2511.2.3 Transfer Using the Trans-Blot Turbo System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2611.2.4 Semi-Dry Transfer Using the Trans-Blot SD Cell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2711.3 Total Protein Blot Stains . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2811.4 Immunodetection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28Chapter 12: Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29Appendix A: Quick Start Guides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31SDS-PAGE (Mini-PROTEAN TGX Gels) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32Native PAGE (Mini-PROTEAN TGX Gels) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33Peptide Analysis (Mini-PROTEAN Tris-Tricine Gels) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 34Nondenaturing Nucleic Acid PAGE (Mini-PROTEAN TBE Gels) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35Denaturing Nucleic Acid PAGE (Mini-PROTEAN TBE-Urea Gels) . . . . . . . . . . . . . . . . . . . . . . . . . . . 36Appendix B: Buffers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37Appendix C: Related Literature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40Appendix D: Ordering Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Mini-PROTEANPrecast Gels 11.1 IntroductionMini-PROTEAN precast gels are 7.2 cm x 8.6 cm gels designed for performing polyacrylamide gelelectrophoresis (PAGE) with the Mini-PROTEAN family of vertical electrophoresis cells, which includesthe Mini-PROTEAN Tetra and Mini-PROTEAN 3 Dodeca cells and the discontinued Mini-PROTEAN IIand Mini-PROTEAN 3 cells. The Mini Trans-Blot , Trans-Blot Turbo , and Trans-Blot SD blotting cellsand precut membrane sandwiches are also available for blotting applications with these gels.Features of Mini-PROTEAN precast gels include:nOutlined and numbered well that simplify sample loading and identificationnCapacity for up to 15 samples per gelnBottom-open cassette design for easy gel handling and blotting setupnEasy-to-open cassette for faster downstream processingnReference line at the bottom of the cassette indicates where the run should stop(for optimum resolution across the separation range)nExcellent staining quality and transfer efficiencynNo gel foot to remove prior to blottingnMini-PROTEAN TGX Stain-Free formulations, which enable rapid 5 min gel imagingwithout staining and destainingComb (available in a range of options)Numbered well outlinesReference line for monitoringprogress of the runTechnical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com1
Mini-PROTEAN Precast Gels1.2 Gel FormulationsMini-PROTEAN precast gels are composed of polyacrylamide with a bisacrylamide crosslinker, and theyare available in a range of formulations (Table 1.1) and in a selection of single percentages and gradients.Table 1.1. Mini-PROTEAN precast gel formulations.ApplicationGel FormulationSample BufferRunning BufferSDS-PAGEMini-PROTEAN TGX Mini-PROTEAN TGX Stain-FreeLaemmliTris/glycine/SDSNative PAGEMini-PROTEAN TGXMini-PROTEAN TGX Stain-FreeNativeTris/glycinePeptide analysisMini-PROTEAN Tris-TricineTricineTris/Tricine/SDSdsDNA separationMini-PROTEAN TBENucleic acidTris/boric acid/EDTA (TBE)ssDNA and RNAseparationMini-PROTEAN TBE-ureaTBE-ureaTBE1.3 Comb ConfigurationsComb TypeWell Volume10-well 50 μl10-well30 μl12-well 20 μl15-well15 μl8 1 well*30 μlIPG/prep7 cm ReadyStrip IPG strip (450 μl)1.4 SpecificationsGel materialPolyacrylamideGel dimensions7.2 x 8.6 cmGel thickness1.0 mmResolving gel height6.2 cm (5.6 cm for 50 μl well)Cassette dimensions8.5 x 10 cmCassette materialStyrene copolymerComb materialPolycarbonateRunning buffer750 ml for 1–2 gels, 1,000 ml for 3–4 gels (Mini-PROTEAN Tetra cell)325 ml for 1–2 gels (Mini-PROTEAN II or Mini-PROTEAN 3 cell)*2Multichannel pipet compatible.Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com
Instruction Manual and Application Guide1.5 Storage ConditionsTable 1.2. Storage conditions for Mini-PROTEAN precast gels. Store gels flat. Shelf life is from date of manufacture;expiration dates are printed on the packaging.StorageTemperatureGel Formulation2–8 CMini-PROTEAN TGX12 monthsMini-PROTEAN TGX Stain-Free12 monthsShelf LifeMini-PROTEAN Tris-Tricine12 weeksMini-PROTEAN TBE12 weeksMini-PROTEAN TBE-urea8 weeks1.6 Important NotesUse each Mini-PROTEAN precast gel as soon as possible after removing it from the storage pouch.Improper storage of Mini-PROTEAN precast gels can produce artifacts. Store gels flat and at 2–8 C.Avoid freezing or prolonged storage above 8 C. If your gels have been stored improperly, discard them.Do not run more than one gel type in the same apparatus at the same time. Different gel percentagesand formulations have different conductivities and different run times.With the Mini-PROTEAN Tetra cell:nWhen running 1–2 gels:Use the electrode assembly (with banana plugs), not the companion running module(without banana plugs)Do not place the companion running module in the tank. Doing so generatesexcessive heat and degrades the quality of the electrophoretic separationnWhen running 3–4 gels, use both the electrode assembly and companion running modulenWhen using voltages 200 V, fill the outer buffer chamber to the 4 gel (800 ml) markTechnical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com3
2Setup and BasicOperation2.1 Workflow OverviewPrepare BuffersPrepare sample and running buffersPrepare Gels andAssemble Electrophoresis CellPrepare and Load SamplesDilute in sample bufferPerform ElectrophoresisSDS-PAGE (Chapter 3)Native PAGE (Chapter 4)Peptide Analysis (Chapter 6)Nondenaturing Nucleic Acid PAGE (Chapter 7)Denaturing Nucleic Acid PAGE (Chapter 8)2-D Electrophoresis (Chapter 9)Analyze the Separation(Chapter 10)Blot the Gels (Optional)(Chapter 11)4Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com
Instruction Manual and Application Guide2.2 Required MaterialsnnnnnnMini-PROTEAN precast gelsMini-PROTEAN Tetra cell (or Mini-PROTEAN 3 Dodeca , Mini-PROTEAN II orMini-PROTEAN 3 cell)PowerPac Basic or PowerPac HC power supply (or equivalent); PowerPac HV orPowerPac Universal required for high-voltage applications ( 300 V)Sample bufferRunning buffer (750 ml for 1–2 gels; 1,000 ml for 3–4 gels or when running atvoltages 200 V)Opening lever (catalog #456-0000)2.3 Setting Up and Running Mini-PROTEAN Gels in theMini-PROTEAN Tetra Cell1.Remove the gels from the storage pouch and prepare them for assembly:a.Remove the comb: Position thumb on the indentation (middle of comb) and remove the combby pulling upward in one smooth motion.b. Remove the tape: Pull gently to remove the green tape from the bottom of the cassette.If necessary, use the opening key or comb to help remove the tape at the corners.c.Rinse the wells: Use a syringe, wash bottle, or disposable transfer pipet to rinse the wells withRemove the combRemove the tapeTechnical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com5
Mini-PROTEAN Precast Gelsrunning buffer. Straighten the sides of thewells, if necessary.2.ASet the electrode assembly to the openposition on a clean, flat surface (A).3. Place the gel cassettes into the electrodeassembly. Two cassettes are required to createa functioning assembly; when using 1 or 3gels, use the buffer dam (included with the cell)to complete the assembly.a.Place the first cassette with the short platefacing inward and so the gel rests at a 30 angle away from the center of the electrodeassembly. Make sure the electrode assemblyremains balanced and does not tip over.Bb. Place the second gel or buffer dam on theother side of the electrode assembly, againby resting the gel on the supports. The gelsrest at 30 angles, one on either side of theelectrode assembly, tilting away from thecenter of the frame (B).4. Gently push both gels toward each other,making sure that they rest firmly and squarelyagainst the green gasket that is built into theelectrode assembly. Align the short plates toensure the edge sits just below the notch atthe top of the green gasket (C).5. While gently squeezing the gel cassettes(or cassette and buffer dam) against the greengaskets (maintaining constant pressure andwith both gels in place), slide the green armsof the clamping frame one at a time over thegels, locking them into place (D,E).Clamping frameCGasketNotchGel cassetteShort plateLong plateGel support6. The wing clamps of the electrode assembly lifteach gel cassette up against the notch in thegreen gasket, forming a seal. Check again thatD6Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.comE
Instruction Manual and Application Guidethe short plates sit just below the notch at the top ofthe green gasket (C).FIf running more than 2 gels, repeat steps 2–6 with thecompanion running module.7.Place the electrophoresis module into the tank (F) andfill the buffer chambers with 1x running buffer:nn200 ml in the inner buffer chamber550 ml (1–2 gels) or 800 ml (3–4 gels, or 200 V) in the outer buffer chamber8. Wash the sample wells with running buffer (if this wasnot done earlier).9. Load samples and run the gels using the runningconditions appropriate to your application. Stop the run when the dye front reaches the referenceline imprinted on the bottoms of the cassettes.2.4 Removing the Gel1.After electrophoresis is complete, turn off the power supply and disconnect the electrical leads.2.Remove the lid from the tank and remove the gels from the cell. Pour off and discard the runningbuffer.3. To open the cassette, align the arrow on the opening lever with the arrows marked on the cassetteand insert the lever between the cassette plates at indicated locations. Apply downward pressure tobreak each seal. Do not twist the lever.4. Pull the two plates apart from the top of the cassette, and gently remove the gel.Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com7
3SDS-PAGE3.1 IntroductionMini-PROTEAN TGX (Tris-Glycine eXtended shelf life) gels provide a versatile system for separatingproteins by either molecular weight (SDS-PAGE) or mass-to-charge ratio (native PAGE). (See Chapter4 for native PAGE applications and protocols.) This versatility is possible because the gels are madewithout SDS; this allows the sample buffer and running buffer to determine the separation mechanism.SDS-PAGE relies on a discontinuous buffer system. Two ions differing in electrophoretic mobility(glycinate and chloride) form a moving boundary when voltage is applied. Proteins have anintermediate mobility that causes them to concentrate, or stack, into a narrow zone at the beginningof electrophoresis. As that zone moves through the gel, the sieving effect of the polyacrylamide gelmatrix causes proteins of different molecular weighs to move at different rates. This stacking effect isresponsible for the high resolving power of SDS-PAGE: the sample is loaded in a relatively broad zone,and the moving boundary concentrates the proteins into sharp bands prior to separation.Protein samples for SDS-PAGE are prepared using SDS and a thiol reducing agent, usuallyβ-mercaptoethanol or dithiothreitol (DTT). SDS forms complexes with proteins, giving them a rodlikeshape and similar mass-to-charge ratio. The reducing agent disrupts disulfide bonds between andwithin proteins, allowing complete denaturation and dissociation. Heat treatment in the presence ofSDS and reducing agent effectively eliminates the effects of native charge and higher order structure onelectrophoretic mobility, so the migration distance depends primarily on molecular weight.Molecular weight is estimated by plotting the logarithm of protein molecular weight vs. the relativemobility (Rf ) of the protein (Rf distance migrated by the protein/distance migrated by the dye front)or by using the point-to-point semilog interpolation method in Quantity One or Image Lab software.Refer to bulletins 3133, 3144, and 10014472 for more information.3.2 Mini-PROTEAN TGX andMini-PROTEAN TGX Stain-Free GelsMini-PROTEAN TGX gels are Laemmli-like gels that have a proprietary modification that extends shelflife to 12 months and enhances separation characteristics relative to conventional gel types. They arerun using standard Laemmli sample buffer and Tris/glycine/SDS running buffer, and they generateprotein migration patterns that are similar to those observed with standard Laemmli Tris-HCl gels.Two types of TGX formulations are available:nn8Mini-PROTEAN TGX — Laemmli-like, extended shelf life gelsMini-PROTEAN TGX Stain-Free — Laemmli-like, extended shelf life gels with trihalocompounds that allow rapid fluorescent detection of proteins with the stain-free system,eliminating staining and destaining steps for faster results (see Chapter 5 for more details)Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com
Instruction Manual and Application GuideBoth gel types gels are available in polyacrylamide single percentages and gradients. Use the proteinmigration charts and tables to select the gel type that optimizes resolution of your sample:nnUse single-percentage gels to separate bands of similar molecular weight. Optimum separationoccurs in the lower half of the gel, so use a percentage in which the protein migrates to thelower half of the gelUse gradient gels to separate samples containing a broad range of molecular weights. Gradientgels allow resolution of both high- and low-molecular weight bands on the same gel. Largerpore sizes at the top of the gel permit resolution of larger molecules, smaller pore sizes towardthe bottom of the gel restrict excessive separation of small moleculesGel CompositionCrosslinker2.6% CStacking gel4% T, 2.6% CShelf life 12 months at 2–8 C; expiration date is printed on packageGel PercentageOptimum SeparationGel PercentageOptimum SeparationRange Range7.5%40–200 kD4–15%20–250 kD10%30–150 kD4–20%10–200 kD12% 20–120 kD Any kD 10–100 kDMini-PROTEAN TGX Precast Gels Broad Range UnstainedPrecision Plus Protein Unstained %250250150150Any kD 45313121.53121.515102004566204515Any kD 6.514.46.514.46.5Migration charts for protein standards on Mini-PROTEAN TGX and Mini-PROTEAN TGX Stain-Free gels.Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com9
Mini-PROTEAN Precast Gels3.3 SDS-PAGE BuffersSee Appendix B for buffer formulations. Do not adjust pH.Running buffer (1x)25 mM Tris, 192 mM glycine, 0.1% SDSDilute 100 ml 10x stock (catalog #161-0732) with 900 ml deionized water (diH2O).Sample buffer (2x)62.5 mM Tris-HCl, pH 6.8, 2% SDS, 25% (v/v) glycerol, 0.01% bromophenolblue, 5% β-mercaptoethanol or 100 mM DTT (added fresh)Use Laemmli sample buffer (catalog #161-0737) and add β-mercaptoethanol orDTT before use.Sample buffer (4x)250 mM Tris-HCl, pH 6.8, 4% LDS, 40% (w/v) glycerol, 0.02% bromophenolblue, 15% beta-mercaptoethanol or 200 mM DTT (added fresh)Use 4x Laemmli sample buffer (catalog #161-0747) and add β-mercaptoethanolor DTT before use.3.4 Sample Preparation1.Determine the appropriate concentration of sample to load (depends on the load volume and thedetection method used; see Chapter 10 for approximate stain sensitivities).2.Dilute the sample with sample buffer with added reducing agent.2x: dilute 1 part sample with 1 part sample buffer.4x: dilute 3 parts sample with 1 part sample buffer.For nonreducing conditions, omit the reducing agent.3. Heat the diluted sample at 90–95 C for 5 min or at 70 C for 10 min.3.5 Running ConditionsRun conditions and times are approximate. Run times represent the time required for the dye frontto reach the line at the bottom of the cassette. Conditions may vary depending on water and bufferconductivity, which vary from one lab setting to the next. Multiply current by the number of gels run.Table 3.1. Standard running conditions for SDS-PAGE in the Mini-PROTEAN Tetra cell.GelOptimum RangeRun Conditions7.5% 40–200 kD10% 30–150 kD300 V constant:12% 20–120 kDStarting current (per gel): 55–75 mA4–15% 20–250 kDFinal current (per gel): 45–70 mA4–20% 10–200 kDAny kD 10–100 kD10Technical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.comRun Time15–20 min(Fill outer buffer volumeto the 4-gel mark)
Instruction Manual and Application GuideTable 3.2. Alternative running conditions for SDS-PAGE in the Mini-PROTEAN Tetra cell.100 V200 V85–95 min30–40 minInitial15–20 mA25–50 mAFinal5–10 mA20–31 mA25 C25–35 C2-gel mark4-gel mark2-gel mark4-gel markRun timeExpected current (per gel)Expected temperatureOuter buffer volume1–2 Gels3–4 GelsTable 3.3. PowerPac power supply recommendations.# Gels100 V200 V300 chnical Support: 1-800-4BIORAD 1-800-424-6723 www.bio-rad.com11
4Native PAGE4.1 In
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BELLA 17234 16 Cup Rice Cooker Instruction Manual October 12, 2021October 24, 2021 Home » Bella » BELLA 17234 16 Cup Rice Cooker Instruction Manual 16 CUP RICE COOKER Instruction Manual and Recipe Guide WWW.BELLAHOUSEWARES.COM Register your product and get support Registrar y obtener asistencia de su producto THANK YOU for your purchase
