Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S Gerstein ISBNS:0-471-37972-7( Paper);0-471 (Electronic) 3 Western Blotting Peter riis Physical Properties of Proteins What do you know about your protein? ..374 What Other Physical Properties Make Your Protein Unusual? ..374 Choosing a Detection Strategy: Overview of Detection Sys 375 What Are the Criteria for Selecting a Detection Method ..377 What Are the Keys to Obtaining High-Quality Results?.. 379 Which Transfer Membrane Is Most Appropriate to our needs Blocking 380 Which Blocking Agent Best Meets Your Needs? Washing ..382 What Composition of Wash Buffer Should You Use? What Are Common Blot Size, Format, and Handling The Primary Antibody Are All Antibodies Suitable for Blotting? How Should antibodies be handled and stored ..384 econdary reagents∴ Secondary Reagent g How Im Is sp Why Are Some Secondary Antibodies Offered as F(ab)2 Amplification ..387 373
373 13 Western Blotting Peter Riis Physical Properties of Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . 374 What Do You Know about Your Protein? . . . . . . . . . . . . . . 374 What Other Physical Properties Make Your Protein Unusual? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 374 Choosing a Detection Strategy: Overview of Detection Systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 375 What Are the Criteria for Selecting a Detection Method? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377 What Are the Keys to Obtaining High-Quality Results? . . . 379 Which Transfer Membrane Is Most Appropriate to Your Needs? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 379 Blocking . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 380 Which Blocking Agent Best Meets Your Needs? . . . . . . . . . . 381 Washing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382 What Composition of Wash Buffer Should You Use? . . . . . 382 What Are Common Blot Size, Format, and Handling Techniques? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 382 The Primary Antibody . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 383 Are All Antibodies Suitable for Blotting? . . . . . . . . . . . . . . . . 383 How Should Antibodies Be Handled and Stored? . . . . . . . . 384 Secondary Reagents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 384 How Important Is Species Specificity in Secondary Reagents? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Why Are Some Secondary Antibodies Offered as F(ab’)2 Fragments? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 385 Amplification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 387 Molecular Biology Problem Solver: A Laboratory Guide. Edited by Alan S. Gerstein Copyright © 2001 by Wiley-Liss, Inc. ISBNs: 0-471-37972-7 (Paper); 0-471-22390-5 (Electronic)
Will the Stripping Procedure Affect the Target Protein ..388 Stripping and Reprobing Can the Same Stripping Protocols Be Used for Membranes from different manufacturers? Is It Always Necessary to Strip a Blot before Troubleshooting 389 Setting Up a New Method Bibliography 397 PHYSICAL PROPERTIES OF PROTEINS What Do You know about Your protein? In order to make informed choices among the bewildering range of available transfer and detection methods, it is best to have as clear an idea as possible of your own particular requirements. In large part these choices will depend on the nature of your target protein Even limited knowledge can be used to advantage How abundant is your protein? It isnt necessary to answer the question in rigorously quantitative terms: an educated guess is suf ficient. Are your samples easy to obtain and plentiful, or limited and precious? Is the sample likely to be rich in target protein(e. g, if the protein is overexpressed)or poor in target(perhaps a cytokine)? Obviously low protein concentration or severely limited sample size would require a more sensitive detection method What is the molecular weight of your target protein? Low MW proteins(12kDa or less) are retained less efficiently than higher molecular weight proteins. Membranes with a pore size of 0.1 or 0.2 micron are recommended for transfer of these smaller pro teins, and PVDF will tend to retain more low Mw protein than nitrocellulose. The ultimate lower limit for transfer is somewhere around 5kDa, although this depends largely on the proteins shape and charge The transfer of high molecular weight proteins(more than 100kDa)can benefit from the addition of up to 0. 1% SDs to the transfer buffer(Lissilour and Godinot, 1990). Transfer time can also be increased to ensure efficient transfer of high molecular ight proteins. What Other Physical Properties Make Your Protein unusual? In cases where proteins are highly basic(where the pl of the protein is higher than the pH of the transfer buffer) the protein 374 Riis
Stripping and Reprobing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 Will the Stripping Procedure Affect the Target Protein? . . . 388 Can the Same Stripping Protocols Be Used for Membranes from Different Manufacturers? . . . . . . . . . . . 389 Is It Always Necessary to Strip a Blot before Reprobing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Setting Up a New Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 PHYSICAL PROPERTIES OF PROTEINS What Do You Know about Your Protein? In order to make informed choices among the bewildering range of available transfer and detection methods, it is best to have as clear an idea as possible of your own particular requirements. In large part these choices will depend on the nature of your target protein. Even limited knowledge can be used to advantage. How abundant is your protein? It isn’t necessary to answer the question in rigorously quantitative terms: an educated guess is suf- ficient.Are your samples easy to obtain and plentiful,or limited and precious? Is the sample likely to be rich in target protein (e.g., if the protein is overexpressed) or poor in target (perhaps a cytokine)? Obviously low protein concentration or severely limited sample size would require a more sensitive detection method. What is the molecular weight of your target protein? Low MW proteins (12kDa or less) are retained less efficiently than higher molecular weight proteins. Membranes with a pore size of 0.1 or 0.2 micron are recommended for transfer of these smaller proteins, and PVDF will tend to retain more low MW protein than nitrocellulose. The ultimate lower limit for transfer is somewhere around 5kDa, although this depends largely on the protein’s shape and charge. The transfer of high molecular weight proteins (more than 100kDa) can benefit from the addition of up to 0.1% SDS to the transfer buffer (Lissilour and Godinot, 1990). Transfer time can also be increased to ensure efficient transfer of high molecular weight proteins. What Other Physical Properties Make Your Protein Unusual? In cases where proteins are highly basic (where the pI of the protein is higher than the pH of the transfer buffer) the protein 374 Riis
will not be carried toward the anode, since transfer takes place on the basis of charge. In these cases it is best to include SDs in the transfer buffer. Alternatively, the transfer sandwich can be assem bled with membranes on both sides of the gel CHOOSING A DETECTION STRATEGY OVERVIEW OF DETECTION SYSTEMS Detection systems range from the simplest colorimetric systems for use on the benchtop to complex instrument-based systems Table 13. 1). The simplest is radioactive detection: a secondary reagent is labeled with a radioactive isotope, usually the low- energy gamma-emitter iodine-125. After the blot is incubated with the primary antibody, the labeled secondary rotein A, but it can be a secondary antibody) is applied, the blot Table 13.1 Comparison of Detection Methods Method Features Limitations Radioactive Can quantitate Use of radioactive lpg material can be densitometry; difficult and expensive obe bl requ no enzymatic licensing radiat training Colorimetric Easy to perform; Relatively 200pg hard cop entitiy results directly on blot. for facilities and equipment Chemiluminescent Highly sensitive; Requires careful l pg(luminol) an quantitate optimization using film 0.1 pg(acridan) metry, Fluorescent Good linear ange for stringent data stored membrane digit d possible but difficult Western blotting 375
will not be carried toward the anode, since transfer takes place on the basis of charge. In these cases it is best to include SDS in the transfer buffer. Alternatively, the transfer sandwich can be assembled with membranes on both sides of the gel. CHOOSING A DETECTION STRATEGY: OVERVIEW OF DETECTION SYSTEMS Detection systems range from the simplest colorimetric systems for use on the benchtop to complex instrument-based systems (Table 13.1). The simplest is radioactive detection: a secondary reagent is labeled with a radioactive isotope, usually the lowenergy gamma-emitter iodine-125.After the blot is incubated with the primary antibody, the labeled secondary reagent (usually Protein A, but it can be a secondary antibody) is applied, the blot Western Blotting 375 Table 13.1 Comparison of Detection Methods Method Features Limitations Sensitivity Radioactive Can quantitate Use of radioactive 1 pg through film material can be densitometry; difficult and can strip and expensive; reprobe blots; requires no enzymatic licensing and development radiation safety step training Colorimetric Easy to perform; Relatively 200 pg hard copy insensitive results directly on blot; minimal requirements for facilities and equipment Chemiluminescent Highly sensitive; Requires careful 1 pg (luminol) can quantitate optimization using film 0.1 pg (acridan) densitometry; can strip and reprobe Fluorescent Good linear Equipment 1 pg range for expensive; quantitation; stringent data stored membrane digitally requirements; stripping and reprobing possible but difficult
washed and exposed to film for hours or days. Radioactive blots can more quickly be detected using storage phosphor plates instead of film; the plates are read on a specialized scanning instru- ment. detailed discussions about the features and benefits of detection by film and scanners are included in Chapter 14, Nucleic Acid Hybridization to indicate the presence of bound antibody The simplest st Enzymatic reactions are used in a number of different systems enzymatic detection is chromogenic. Here the secondary reagent is conjugated to an enzyme, either horseradish peroxidase (HRP) or alkaline phosphatase(AP). After incubation with the sec- ondary reagent and washing, the blot is incubated with a substrate. The enzyme catalyzes a reaction in which the substrate is con- verted to a colored precipitate directly on the membrane, essen tially coloring the band on which the primary antibody has bound While not as sensitive as other methods colorimetric detection is fast and simple, and requires no special facilities. Chemiluminescent detection combines characteristics of both radioactive and chromogenic detection. Again, an enzyme label is used(commonly HRP, but there are systems for use with AP as well), but in this case the reaction produces light rather than a colored product as a result of reaction. The light is usually cap tured on X-ray film, just like a radioactive blot. Specialized imaging equipment for chemiluminescent blots is also available Chemiluminescent detection is very sensitive, and the blots are easily stripped for subsequent reprobing There are significant differences in the various available chemi luminescent detection systems. The most widely used are the luminol-based HRP systems. These typically emit usable signals for an hour or two. There are also newer, higher-sensitivity HRP- based systems that emit light for more than 24 hours; however, these substrates are more expensive and require even more careful optimization than the luminol-based systems. AP-based hemiluminescent systems are also available. They are not also emit light for extended periods. Those systems produc- ing extended light output have the advantage that several ex posures can be taken from the same blot With the availability of fluorescence-scanning instruments, new methods for detection have come into use. It may seem at first glance that a secondary antibody could simply be coupled to a flu orescent molecule and the detection performed directly. Although this is possible, this method is not sufficiently sensitive for most purposes. The approach usually taken uses an enzyme-coupled 376 Riis
washed and exposed to film for hours or days. Radioactive blots can more quickly be detected using storage phosphor plates instead of film; the plates are read on a specialized scanning instrument. Detailed discussions about the features and benefits of detection by film and scanners are included in Chapter 14, Nucleic Acid Hybridization. Enzymatic reactions are used in a number of different systems to indicate the presence of bound antibody. The simplest type of enzymatic detection is chromogenic. Here the secondary reagent is conjugated to an enzyme, either horseradish peroxidase (HRP) or alkaline phosphatase (AP). After incubation with the secondary reagent and washing, the blot is incubated with a substrate. The enzyme catalyzes a reaction in which the substrate is converted to a colored precipitate directly on the membrane, essentially coloring the band on which the primary antibody has bound. While not as sensitive as other methods, colorimetric detection is fast and simple, and requires no special facilities. Chemiluminescent detection combines characteristics of both radioactive and chromogenic detection. Again, an enzyme label is used (commonly HRP, but there are systems for use with AP as well), but in this case the reaction produces light rather than a colored product as a result of reaction. The light is usually captured on X-ray film, just like a radioactive blot. Specialized imaging equipment for chemiluminescent blots is also available. Chemiluminescent detection is very sensitive, and the blots are easily stripped for subsequent reprobing. There are significant differences in the various available chemiluminescent detection systems. The most widely used are the luminol-based HRP systems. These typically emit usable signals for an hour or two. There are also newer, higher-sensitivity HRPbased systems that emit light for more than 24 hours; however, these substrates are more expensive and require even more careful optimization than the luminol-based systems. AP-based chemiluminescent systems are also available. They are not widely used in Western blotting, but they are highly sensitive and also emit light for extended periods. Those systems producing extended light output have the advantage that several exposures can be taken from the same blot. With the availability of fluorescence-scanning instruments, new methods for detection have come into use. It may seem at first glance that a secondary antibody could simply be coupled to a fluorescent molecule and the detection performed directly.Although this is possible, this method is not sufficiently sensitive for most purposes. The approach usually taken uses an enzyme-coupled 376 Riis
secondary reagent(in this case usually AP) and a substrate that produces an insoluble, fluorescent product. The enzymatic reac tion results in amplification of the signal, giving much better sen sitivity than a fluorescently tagged secondary reagent. The blot is read on a fluorescent scanner and recorded as a digitized image What Are the Criteria for Selecting a Detection Method? There is a natural tendency to choose the most sensitive method available. High-sensitivity systems are desirable for detection of low-abundance proteins, but they are also desirable in cases where primary antibody is expensive or in limited supply, since these systems allow antibodies to be used at high dilutions. On the other hand, low-sensitivity systems, especially chromogenic systems, are easy to work with, require less exacting optimization, and tend to be less prone to background problems. Sensitivity overkill can be more trouble than it is worth What can you conclude from commercial sensitivity data? It can be difficult to compare the claims of sensitivity made by commercial suppliers. Although there is nothing wrong with the way these values are established, comparison between different systems can be difficult because the values depend on the exact conditions under which the determination was made. The primary antibody has a large effect on the overall sensitivity of any system, so comparisons between systems using different primary antibod ies are less meaningful than they may seem at first glance. In order to compare two different detection systems, the target protein, the primary antibody, and, where possible, the secondary reagent should be the same. Such direct comparisons are hard to come by Also sensitivity claims are usually made with purified protein rather than with crude lysate. For these reasons commercial sen sitivity claims should be considered approximate, and it may be unrealistic to expect to attain the same level of sensitivity in your own system as that quoted by the manufacturer. urat Will your research situation require extended signal output in order to prepare several exposures from the same blot? ability to quantitate Film-based systems(chemiluminescent and radioactive)as well as fluorescence-scanning methods, allow quantitation of target proteins. Results on film are quantified by densitometry, while the Western blotting 377
secondary reagent (in this case usually AP) and a substrate that produces an insoluble, fluorescent product. The enzymatic reaction results in amplification of the signal, giving much better sensitivity than a fluorescently tagged secondary reagent. The blot is read on a fluorescent scanner and recorded as a digitized image. What Are the Criteria for Selecting a Detection Method? Sensitivity There is a natural tendency to choose the most sensitive method available. High-sensitivity systems are desirable for detection of low-abundance proteins, but they are also desirable in cases where primary antibody is expensive or in limited supply, since these systems allow antibodies to be used at high dilutions. On the other hand, low-sensitivity systems, especially chromogenic systems, are easy to work with, require less exacting optimization, and tend to be less prone to background problems. Sensitivity overkill can be more trouble than it is worth. What can you conclude from commercial sensitivity data? It can be difficult to compare the claims of sensitivity made by commercial suppliers. Although there is nothing wrong with the way these values are established, comparison between different systems can be difficult because the values depend on the exact conditions under which the determination was made. The primary antibody has a large effect on the overall sensitivity of any system, so comparisons between systems using different primary antibodies are less meaningful than they may seem at first glance. In order to compare two different detection systems, the target protein, the primary antibody, and, where possible, the secondary reagent should be the same. Such direct comparisons are hard to come by. Also sensitivity claims are usually made with purified protein rather than with crude lysate. For these reasons commercial sensitivity claims should be considered approximate, and it may be unrealistic to expect to attain the same level of sensitivity in your own system as that quoted by the manufacturer. Signal Duration Will your research situation require extended signal output in order to prepare several exposures from the same blot? Ability to Quantitate Film-based systems (chemiluminescent and radioactive) as well as fluorescence-scanning methods, allow quantitation of target proteins. Results on film are quantified by densitometry, while the Western Blotting 377