Bulletin 2895 Protein Blotting Guide

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Transcript of Bulletin 2895 Protein Blotting Guide

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    A Guide to Transfer and DetectionThird Edition

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    Protein Blotting Guide 1

    About This Manual

    This reference manual provides information on the fundamentals of protein transfer and

    detection chemistries. It is also a guide to the methods, equipment, and reagents used

    in protein blotting experiments, and offers troubleshooting tips and technical advice.

    As a researcher using the blotting technique, you know that each research objective,

    experimental approach, and protein sample can be different. Your equipment, sample,

    antibody, and detection chemistries all can impact your results. The goal of this manual is to

    provide you with a broad understanding of the variables you face when blotting, and how

    best to work with each of them to achieve the optimal results.

    A History of Leadership and QualityA pioneer in the design and manufacture of western blotting apparatus with 30 years of

    experience, Bio-Rad is considered the industry leader in providing high-quality, durable, andpowerful blotting equipment.

    Bio-Rad offers superior products and expert technical service. Our goal is to support

    your research with the necessary tools and materials to optimize the analysis of complex

    protein samples.

    Meeting Your Blotting NeedsTurn to Bio-Rad for:

    Gel-blotting equipment for an array of gel sizes

    Microfiltration devices

    Multiple-sample screening devices

    Membranes for every binding requirement

    Protein standards Blotting buffers, reagents, and background removal kits

    Colorimetric and chemiluminescent detection reagents

    Total protein stains

    Secondary antibodies and antibody conjugates

    For detailed protocols on the use of any of the products mentioned in this guide, please refer to

    their instruction manuals, available in Adobe Acrobat (PDF) format at discover.bio-rad.com

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    Introduction to Protein Blotting 7

    Chapter 1 Transfer

    Transfer Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . .9

    Electrophoretic Transfer

    The Principle of Electrophoretic Transfer

    Types of Electrophoretic Transfer

    Microfiltration (Dot-Blotting) . . . . . . . . . . . . . . . . . . . .11

    Blotting Systems and Power Supplies . . . . . . . . . . . .12

    Transfer Apparatus . . . . . . . . . . . . . . . . . . . . . . . . . .12

    Tank Blotting ApparatusSemi-Dry Blotting Apparatus

    Microfiltration Apparatus

    Power Supplies for Electrophoretic Transfers . . . . . . .15

    Chapter 2 Membranes, Buffers, andPower Conditions

    Membrane Selection . . . . . . . . . . . . . . . . . . . . . . . . . .17

    Nitrocellulose and Supported Nitrocellulose . . . . . . . .17

    Polyvinylidene Difluoride (PVDF) Membrane . . . . . . . .18

    Blotting Filter Paper . . . . . . . . . . . . . . . . . . . . . . . . . . .18

    Membrane/Filter Paper Sandwiches . . . . . . . . . . . . . .19

    Transfer Buffer Selection . . . . . . . . . . . . . . . . . . . . . . .19

    General Recommendations . . . . . . . . . . . . . . . . . . . .20A Note About SDS and Alcohol

    Towbin and Bjerrum and Schafer-Nielsen Buffers(Tris/Glycine Buffers) . . . . . . . . . . . . . . . . . . . . . . . . .20

    CAPS Buffer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .21

    Discontinuous Tris-CAPS Buffer System(Semi-Dry Transfers) . . . . . . . . . . . . . . . . . . . . . . . . .21

    Dunn Carbonate Buffer . . . . . . . . . . . . . . . . . . . . . . .21

    Alternative Buffer Conditions . . . . . . . . . . . . . . . . . . .21

    Power Conditions for Electrophoretic Transfers . . . . .21

    Useful Equations . . . . . . . . . . . . . . . . . . . . . . . . . . .22

    Joule Heating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

    Other Factors Affecting Transfer . . . . . . . . . . . . . . . .22Relationship Between Power Settings and

    Transfer Times . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .22

    High-Intensity Field Transfers

    Standard Field Transfers

    Selecting Power Supply Settings . . . . . . . . . . . . . . . .22

    Transfers Under Constant Voltage

    Transfers Under Constant Current

    Transfers Under Constant Power

    General Guidelines for Transfer Buffers

    and Transfer Conditions . . . . . . . . . . . . . . . . . . . . . . .24

    Chapter 3 Performing the Transfer

    Electrophoretic Transfer . . . . . . . . . . . . . . . . . . . . . . .25

    Performing a Tank Transfer . . . . . . . . . . . . . . . . . . . . .25

    Preparing the Transfer Buffer, Gels, andTank Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . .25

    Assembling the Gel and Membrane Sandwich . . . . . .26

    Performing the Transfer . . . . . . . . . . . . . . . . . . . . . .27

    Performing a Semi-Dry Transfer . . . . . . . . . . . . . . . . .27

    Preparing the Transfer Buffer and Gels . . . . . . . . . . .27

    Assembling the Gel and Membrane Sandwich . . . . . .28

    Performing the Transfer . . . . . . . . . . . . . . . . . . . . . . .28

    Microfiltration . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

    Application of the Vacuum . . . . . . . . . . . . . . . . . . . .28

    Proper Drainage . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

    Flow Valve Extended Incubations . . . . . . . . . . . . .29

    Flow Valve Gentle Vacuum . . . . . . . . . . . . . . . . . .29

    Filtering or Centrifugation of Samples . . . . . . . . . . . .29

    Air Bubbles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .29

    Membrane Removal . . . . . . . . . . . . . . . . . . . . . . . . .29

    Protein Blotting Guide 3

    Table of Contents Protein Blotting

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    Chapter 4 Detection

    Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . . .31

    Protein Standards . . . . . . . . . . . . . . . . . . . . . . . . . . . .32

    Prestained Standards for Western Blotting . . . . . . . . .33

    Recombinant Prestained Standards

    Natural Prestained SDS-PAGE Standards

    Unstained Standards for Western Blotting . . . . . . . . .35

    Total Protein Staining . . . . . . . . . . . . . . . . . . . . . . . . .36

    Anionic Dyes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

    Colloidal Gold . . . . . . . . . . . . . . . . . . . . . . . . . . . . .37

    Biotinylation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

    Fluorescence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .38

    Immunological Detection Systems . . . . . . . . . . . . . . .38

    Blocking Reagents . . . . . . . . . . . . . . . . . . . . . . . . . .39

    Antibody Incubations . . . . . . . . . . . . . . . . . . . . . . . .39

    Primary Antibodies

    Species-Specific Secondary Antibodies

    Antibody-Specific Ligands

    Washes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .41

    Detection Methods . . . . . . . . . . . . . . . . . . . . . . . . . .41Colorimetric Detection

    Chemiluminescent Detection

    Other Detection Methods

    Imaging Documentation and Analysis Methods . . .48

    Luminescent Detection

    Fluorescent, Chemifluorescent, andColorimetric Detection

    Autoradiography

    Screening Apparatus . . . . . . . . . . . . . . . . . . . . . . . .50

    Chapter 5 Troubleshooting

    Transfer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .52

    Electrophoretic Transfer . . . . . . . . . . . . . . . . . . . . . .52

    Poor Electrophoretic Transfer

    Swirls or Missing Patterns; Diffuse Transfers

    Gel Cassette Pattern Transferred to Blot

    Poor Binding to the Membrane Nitrocellulose

    Poor Binding to the Membrane PVDF

    Blotting Standards . . . . . . . . . . . . . . . . . . . . . . . . . .55

    Missing Bands

    Molecular Weight Assignments for Natural(Nonrecombinant) Prestained Standards DifferFrom Lot to Lot

    A Proteins Molecular Weight Differs FromExpected Molecular Weight

    Variation in Mobility Between Recombinant andNatural Prestained Standards of the SameMolecular Weight

    Microfiltration Blotting . . . . . . . . . . . . . . . . . . . . . . . .56

    Leakage or Cross-Well Contamination

    Uneven Filtration or No Filtration

    Halos Around the Wells

    Detection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .57

    Immunological Detection . . . . . . . . . . . . . . . . . . . . .57

    Overall High Background

    Nonspecific Reactions Between Bound Proteinsand Probes

    No Reaction or Weak Signal

    Tests for Monitoring Reagent Activity

    Multiscreen Apparatus . . . . . . . . . . . . . . . . . . . . . . .59

    Leakage or Cross-Well Contamination

    Bubbles Trapped Within the Channels

    Halos Around the Wells

    Total Protein Detection . . . . . . . . . . . . . . . . . . . . . . .59

    Colloidal Gold Total Protein Stain High Background

    Colloidal Gold Total Protein Stain Low Sensitivity

    Biotin-BlotTotal Protein Detection High Background

    Biotin-Blot Total Protein Detection No Reaction or Weak Color Development

    Anionic Dyes High Background

    Anionic Dyes Low Sensitivity

    4 Protein Blotting Guide

    Table of Contents Protein Blotting

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    Appendices

    Transfer Buffer Formulations . . . . . . . . . . . . . . . . . . . .62

    Detection Buffer