E D I T O R I A L
Editorial: Antibody Can Get It Right: Confronting Problems of Antibody Specificity and Irreproducibility Agnes Schonbrunn, PhD Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77225
ntibodies are indispensable research reagents. Their use in endocrinology has exploded since the original radioimmunoassays were developed to measure hormone levels. They are now routinely used in molecular endocrinology research to detect and quantitate cellular proteins and determine their distribution in tissues, in cells, and in subcellular compartments. They detect changes in protein structure produced by covalent modification during cell signaling (eg, phosphorylation), transcriptional regulation (acetylation and methylation), or protein degradation (ubiquitination). They identify and detect protein-protein and protein-DNA interactions by coimmunoprecipitation. Antibodies are used as tools to sort cells, to identify drug targets in normal and diseased tissue, and to monitor disease status and progression. And that is just the tip of the iceberg. Unfortunately, the widespread use of antibodies has also generated enormous controversy: the inability of investigators to replicate published data often results from false-positive or false-negative results produced with antibodies that have not been properly validated (1–5). The problems are particularly intense in fields that use antibodies to analyze proteins that are expressed at low levels in cells: G protein– coupled receptors, steroid hormone receptors, ion channels, transporters, and signal-transducing enzymes such as adenylyl cyclase (6 –22). In addition, ultrasensitive detection methods have compounded the problem as illustrated by studies aimed at detecting low-abundance protein–DNA interactions (eg, chromatin immunoprecipitation assays) (5, 23). So what can we do as individual scientists and as journal editors to ensure the reliability and reproducibility of the data that we generate, the data that we publish, and the data that we rely upon to formulate new hypotheses and plan future experiments?
A
doi: 10.1210/me.2014-1230
Challenge 1—Identifying a Reliable Antibody for the Job at Hand Investigators can either purchase antibodies or make their own, and there are major advantages and disadvantages to each choice. However, in either case, validation of antibodies is the responsibility of the user—validation of commercial antibodies by their suppliers is usually inadequate and frequently unreliable. The advantages of purchasing antibodies are obvious. Good antibodies can take months to produce, there are substantial up-front costs, and there is no guarantee that a particular antigen will induce antibodies of the desired properties in immunized animals. The hope is that purchasing antibodies will eliminate production delays, reduce the investment of time and money in a particular experiment, and preclude the possibility of failure to generate the desired reagent. However, identifying an appropriate antibody for purchase is no simple task: there are often dozens of antibodies available to a target protein with little information provided as to their affinity or specificity. One might think that a manufacturer’s catalog number would provide a unique identifier for a particular antibody. However, this is not the case. Vendors usually assign catalog numbers for an antibody based on the immunizing antigen, the manner in which the antibody was produced (host animal, polyclonal or monoclonal, affinity purification, etc.), and the manufacturer that produced it. In the case of polyclonal antibodies, vendors may use the same catalog number not only for different blood collections from an individual immunized animal but also for blood collections from different host animals immunized with the same antigen. Because each blood sample collected from each animal provides a unique combination of antibody clones and concentrations, this practice can result in immense lot-to-lot variability in an-
Mol Endocrinol, September 2014, 28(9):1403–1407
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tibodies. To add further confusion, validation data pro- the nature of the immunizing antigen is kept confidential. vided by a vendor for an antibody may not have been When investigators generate their own polyclonal antigenerated with the current lot of that antibody. Although body, they can produce both preimmune serum and extra monoclonal antibodies would not be expected to show immunizing antigen for subsequent validation experisuch lot-to-lot variability, in fact they can. For example ments. Generating monoclonal antibodies will often yield Pozner-Moulis et al (24) demonstrated that two different multiple hybridoma clones that can be compared to help lots of a monoclonal antibody to the Met tyrosine kinase ensure that any observed reactivity is specific for the tarreceptor showed opposite staining patterns in an array of get protein. Often the decision to buy an antibody or to generate more than 600 breast cancer cases: one showed nuclear and the other membranous and cytoplasmic staining. One one’s own will be determined by whether a commercial final word of caution: a particular antibody may be sold antibody has been demonstrated by others to “work” for by several distributors under different catalog numbers. a desired application and whether the antibody will be used for a short-term or a long-term project. Caveat emptor. Because the documentation provided by manufacturers is often inadequate, a number of searchable databases have been established to inventory and index antibodies Challenge 2—Validating Antibodies from multiple vendors and to list publications that have Once one or more promcited each antibody (for exising antibodies are amples, see Refs. 25–29). “It is the user’s responsibility to identified, whether purSuch databases are very helpensure that these antibodies are chased or generated inful: articles that carefully sufficiently sensitive and specific to house, it is the user’s revalidate an antibody for a produce reliable results in the sponsibility to ensure specific application often prothat these antibodies are vide the most useful guide for proposed studies. The literature sufficiently sensitive antibody selection, and au- documenting the many problems and specific to produce thors who generate such in- and incorrect conclusions resulting reliable results in the formation ought to be com- from poorly validated antibodies proposed studies. The mended and cited. However, shows that vigorous skepticism and literature documenting it is important to remember critical analysis are a must during the many problems and that the results in such articles incorrect conclusions this process (1–18).” may have been produced usresulting from poorly ing a lot that is no longer validated antibodies shows that vigorous skepticism and available. Unfortunately, many published articles do not provide catalog numbers or lot numbers for the antibod- critical analysis are a must during this process (1–18). All ies used and hence cannot be included in these databases. antibodies cross-react to some extent (1, 30). In any parBecause commercial antibodies often lack the sensitiv- ticular situation, the ratio of specific to nonspecific bindity and specificity required for an application, user vali- ing to target and off-target reactors will be determined by dation is essential. In fact, testing of purchased antibodies the relative abundance of each of the reactors in a particoften requires a substantial investment of time and ular preparation, the affinity of the antibody for each money, and there is no guarantee that a satisfactory re- reactor, the antibody concentration used in the assay, and agent will be identified for the intended use or that it will the method of sample preparation. Off-target reactivity continue to be available. When investigators choose to with polyclonal antibodies can be produced by any of the generate their own antibodies, they do so to ensure that antibody species present in the preparation. However, the work invested in antibody validation is not wasted monoclonal antibodies also show nonspecific reactivity and will result in sufficient amounts of a carefully char- (31). In fact, because mouse monoclonal antibodies tend acterized reagent to complete a study. However, there are to have lower binding affinities than rabbit polyclonal also some useful side benefits. Investigators can control antibodies and are often used at high concentrations, nonthe purity of the antigen they use for immunization, specific binding can be a prohibitive problem. A large number of thoughtful articles provide detailed whether it is a chemically synthesized peptide or a purified protein, thus helping to ensure that the resulting antibod- guidance for proper antibody validation and discuss the ies will be specific. Vendors normally do not disclose the limitation and interpretation of each type of control (13, purity of their immunogens, and, in many instances, even 30 –36). Investigators who use antibodies need to be fa-
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miliar with this literature and rigorously apply the criteria antibody produces a “clean” Western blot with no signal, recommended. It is particularly important to remember implying a lack of sensitivity for the target protein, but that the reactivity of an antibody with a target protein rather that many bands are observed on immunoblots, depends on the concentration of that protein in the prep- none of which correspond to the target protein; ie, the aration. Thus, the fact that an antibody is able to detect a antibody is both insensitive and lacks specificity. Alprotein at the high levels observed in transfected cell lines though it is understandable that antibodies produced to does not guarantee that it will do so in nontransfected native proteins may not recognize the corresponding decells or tissues. As a result, the most stringent control for natured protein after SDS-PAGE, this is not true for anantibody specificity requires comparison of antibody re- tibodies produced against short peptides. Thus, rather activity in wild-type tissues or cells to reactivity in knock- than being reassuring, a vendor’s statement that an antiout animals or cell lines in which endogenous protein peptide antibody does not work on immunoblots should expression has been silenced. For human tissue samples be cause for concern. We as well as others (24) have found in which such a knockout control is not feasible, an that immunoblotting provides a more sensitive and disalternate stringent control would be the use of multiple criminating method for antibody validation than immuantibodies recognizing different epitopes in the same nocytochemistry because it so clearly distinguishes crossreacting proteins of different molecular sizes. Moreover, target protein. Some common misconceptions have particularly im- it is far more likely that an antipeptide antibody will peded reliable antibody usage. A control frequently used “work” in Western blotting and not in immunocytoto evaluate antibody specificity is to determine the effect chemistry than the reverse. The only exception that we that preincubation with excess antigen has on antibody have encountered occurred when only a very small fraction of cells in a tissue exreactivity: the antigen pressed the target protein. In preadsorption test (31). “The most stringent control for that circumstance, the target Although this control protein constitutes such a can sometimes be use- antibody specificity requires small part of a tissue extract ful, it is certainly not comparison of antibody reactivity that it is not detectable in an sufficient. Blocking re- in wild-type tissues or cells to immunoblot. Thus, all antiactivity with excess an- reactivity in knockout animals or peptide antibodies should be tigen demonstrates that cell lines in which endogenous tested by immunoblotting tisthe reactivity is prosues and/or cells that do and duced by an antibody protein expression has been silenced. For human tissue samples do not contain the target prothat recognizes that antein to ensure (a) that the intigen. However, it does in which such a knockout control is tact protein is detected with not demonstrate that not feasible, an alternate stringent the necessary sensitivity and the antibody is specific control would be the use of (b) that reactivity with offonly for that antigen. In multiple antibodies recognizing target proteins is minimal. fact, because this condifferent epitopes in the same Although extensive validatrol is often carried out tion of many commonly used using very high concen- target protein.” trations of blocking anantibodies (eg, anti-ERK) is tigen, low-affinity as well as high-affinity antibodies in a no longer required or expected because of the enormous preparation will be blocked, and the binding of all these amount of published data that demonstrate their specificantibodies to both on-target and off-target reactors will ity and provide reliable positive controls, this is not the be inhibited. Thus, although the antigen preadsorption case for antibodies to receptors and many other proteins test can identify the population of antibodies responsible involved in cell signaling. Thus, experiments that test anfor observed reactivity, it cannot demonstrate that those tibody specificity with clear positive and negative controls antibodies are specific. are essential— otherwise an entire project may be based A false sense of security may also be provided by a statement from vendors that their antibody works for on an artifact, and valuable research resources will be immunocytochemistry or immunohistochemistry but wasted when others try to replicate published data (6 – does not work on immunoblots: such a statement pro- 16). Investigators who wish to avoid the embarrassment vides an excuse not to bother with Western blot controls. of incorrect conclusions must adopt the mantra: validate However, this statement usually does not mean that an early and often.
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Challenge 3—Publishing Sufficient Information to Allow Critical Evaluation of Experimental Data and Facilitate Reproducibility Journals have a responsibility to help ensure that the articles they publish report valid and reproducible results. Unfortunately, journals have put increasing emphasis on reducing the length of published articles. Thus, the space devoted to describing materials and methods has been dramatically reduced over the years. The source of many reagents is missing or inadequately described. Critical control experiments have been relegated to “data not shown.” Figures display Western blots with only slivers of gels containing bands of undefined molecular weights. Frequently, only summary graphs, but no original data, are shown. These widespread practices prevent assessment of either the sensitivity or the specificity of the antibodies utilized and thus endanger the conclusions reached. The good news is that the advent of electronic publication now permits rigid space restrictions to be relaxed. Molecular Endocrinology plans to take full advantage of our change to electronic publication to ensure that the articles in our journal are reliable and reproducible. To guarantee that research reagents are clearly identified, we plan to follow the excellent example set by Endocrinology (37) and publish a full description of all antibodies used in a study. Essential information to be provided includes the following: the name of the individual or vendor who supplied the antibody, the immunizing antigen used to generate the antibody, the nature of the antibody preparation (species, polyclonal or monoclonal, and affinity purified or not), the catalog number, and, importantly, the lot number. We encourage authors to include this information in all submitted papers starting immediately and will require this information starting January 1, 2015. In addition, as a service to the research community, we encourage authors to also list antibodies that they have tested and found to be unsatisfactory. Finally, to help ensure that results in our journal are both reliable and reproducible, we commit to publishing the experimental validation for each new antibody utilized (positive and negative controls, full gels for immunoblots showing the molecular weights of all stained bands, etc.). If antibodies have been validated in a previous publication, this must be cited, and the validating experiments and results should be summarized in the text to provide confidence in the present publication. In all instances, investigators need to describe, and preferably show, the experiments demonstrating that their antibodies are specifically detecting the target protein under their
Mol Endocrinol, September 2014, 28(9):1403–1407
experimental conditions. Such supporting data may go into the body of the article or into a supplement, as the authors see fit. Antibodies are extremely powerful and sensitive reagents that provide the foundation on which many different molecular studies are built. However, they are capable of producing not just misleading but completely incorrect results. By following best practices and exercising great caution with a healthy dose of skepticism, we can ensure that published data are both reliable and reproducible and that the antibodies we use “get it right.” Agnes Schonbrunn, PhD Associate Editor
Acknowledgments I am grateful to the many colleagues who offered helpful suggestions and comments on this article. Address all correspondence and requests for reprints to: Agnes Schonbrunn, Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, PO Box 20708, Houston, TX 77225. E-mail: [email protected]. A.S. is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (Grant DK032234-26). Disclosure Summary: The author has nothing to disclose.
References 1. Rhodes KJ, Trimmer JS. Antibodies as valuable neuroscience research tools versus reagents of mass distraction. J Neurosci. 2006; 26:8017– 8020. 2. Pradidarcheep W, Labruye`re WT, Dabhoiwala NF, Lamers WH. Lack of specificity of commercially available antisera: better specifications needed. J Histochem Cytochem. 2008;56:1099 –1111. 3. Couchman JR. Commercial antibodies: the good, bad, and really ugly. J Histochem Cytochem. 2009;57:7– 8. 4. Perkel JM. The antibody challenge. BioTechniques. 2014;56:111– 114. 5. Parseghian MH. Hitchhiker antigens: inconsistent ChIP results, questionable immunohistology data, and poor antibody performance may have a common factor. Biochem Cell Biol. 2013;91: 378 –394. 6. Lu X, Bartfai T. Analyzing the validity of GalR1 and GalR2 antibodies using knockout mice. Naunyn-Schmiedebergs Arch Pharmacol. 2009;379:417– 420. 7. Bodei S, Arrighi N, Spano P, Sigala S. Should we be cautious on the use of commercially available antibodies to dopamine receptors? Naunyn-Schmiedebergs Arch Pharmacol. 2009;379:413– 415. 8. Jensen BC, Swigart PM, Simpson PC. cTen commercial antibodies for ␣-1-adrenergic receptor subtypes are nonspecific. NaunynSchmiedebergs Arch Pharmacol. 2009;379:409 – 412. 9. Hamdani N, van der Velden J. Lack of specificity of antibodies directed against human -adrenergic receptors. Naunyn-Schmiedebergs Arch Pharmacol. 2009;379:403– 407. 10. Pradidarcheep W, Stallen J, Labruye`re WT, Dabhoiwala NF, Michel MC, Lamers WH. Lack of specificity of commercially available
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antisera against muscarinergic and adrenergic receptors. NaunynSchmiedebergs Arch Pharmacol. 2009;379:397– 402. Kirkpatrick P. Specificity concerns with antibodies for receptor mapping. Nat Rev Drug Discov. 2009;8:278. Pyke C, Knudsen LB. The glucagon-like peptide-1 receptor— or not? Endocrinology. 2013;154:4 – 8. Michel MC, Wieland T, Tsujimoto G. How reliable are G-proteincoupled receptor antibodies? Naunyn-Schmiedebergs Arch Pharmacol. 2009;379:385–388. Antoni FA, Wiegand UK, Black J, Simpson J. Cellular localisation of adenylyl cyclase: a post-genome perspective. Neurochem Res. 2006;31:287–295. Jositsch G, Papadakis T, Haberberger RV, Wolff M, Wess J, Kummer W. Suitability of muscarinic acetylcholine receptor antibodies for immunohistochemistry evaluated on tissue sections of receptor gene-deficient mice. Naunyn-Schmiedebergs Arch Pharmacol. 2009;379:389 –395. Everaerts W, Sepu´lveda MR, Gevaert T, Roskams T, Nilius B, De Ridder D. Where is TRPV1 expressed in the bladder, do we see the real channel? Naunyn-Schmiedebergs Arch Pharmacol. 2009;379: 421– 425. Drucker DJ. Incretin action in the pancreas: potential promise, possible perils, and pathological pitfalls. Diabetes. 2013;62:3316 – 3323. Zhou Y, Waanders LF, Holmseth S, Guo C, et al. Proteome analysis and conditional deletion of the EAAT2 glutamate transporter provide evidence against a role of EAAT2 in pancreatic insulin secretion in mice. J Biol Chem. 2014;289:1329 –1344. Welsh AW, Lannin DR, Young GS, et al. Cytoplasmic estrogen receptor in breast cancer. Clin Cancer Res. 2012;18:118 –126. Grimsey NL, Goodfellow CE, Scotter EL, Dowie MJ, Glass M, Graham ES. Specific detection of CB1 receptors; cannabinoid CB1 receptor antibodies are not all created equal! J Neurosci Methods. 2008;171:78 – 86. Snyder MA, Smejkalova T, Forlano PM, Woolley CS. Multiple ER antisera label in ER knockout and null mouse tissues. J Neurosci Methods. 2010;188:226 –234. Herrera M, Sparks MA, Alfonso-Pecchio AR, Harrison-Bernard LM, Coffman TM. Lack of specificity of commercial antibodies leads to misidentification of angiotensin type 1 receptor protein. Hypertension. 2013;61:253–258. Mechetner L, Sood R, Nguyen V, Gagnon P, Parseghian MH. The
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effects of hitchhiker antigens co-eluting with affinity-purified research antibodies. J Chromatogr B Analyt Technol Biomed Life Sci. 2011;879:2583–2594. Pozner-Moulis S, Cregger M, Camp RL, Rimm DL. Antibody validation by quantitative analysis of protein expression using expression of Met in breast cancer as a model. Lab Invest. 2007;87:251– 260. Helsby MA, Leader PM, Fenn JR, et al. CiteAb: a searchable antibody database that ranks antibodies by the number of times they have been cited. BMC Cell Biol. 2014;15:6. Marx V. Finding the right antibody for the job. Nat Methods. 2013;10:703–707. Helsby MA, Fenn JR, Chalmers AD. Reporting research antibody use: how to increase experimental reproducibility. F1000Research. 2013;2:153. The Antibody Registry. http://antibodyregistry.org/. Accessed August 1, 2014. Bjo¨rling E, Uhle´n M. Antibodypedia, a portal for sharing antibody and antigen validation data. Mol Cell Proteomics. 2008;7:2028 – 2037. Fritschy JM. Is my antibody-staining specific? How to deal with pitfalls of immunohistochemistry. Eur J Neurosci. 2008;28:2365– 2370. Holmseth S, Zhou Y, Follin-Arbelet VV, Lehre KP, Bergles DE, Danbolt NC. Specificity controls for immunocytochemistry: the antigen preadsorption test can lead to inaccurate assessment of antibody specificity. J Histochem Cytochem. 2012;60:174 –187. Saper CB, Sawchenko PE. Magic peptides, magic antibodies: guidelines for appropriate controls for immunohistochemistry. J Comp Neurol. 2003;465:161–163. Lorincz A, Nusser Z. Specificity of immunoreactions: the importance of testing specificity in each method. J Neurosci. 2008;28: 9083–9086. Saper CB. A guide to the perplexed on the specificity of antibodies. J Histochem Cytochem. 2009;57:1–5. Bordeaux J, Welsh A, Agarwal S, et al. Antibody validation. BioTechniques. 2010;48:197–209. Ivell R, Teerds K, Hoffman GE. Proper application of antibodies for immunohistochemical detection: antibody crimes and how to prevent them. Endocrinology. 2014;155:676 – 687. Gore AC. Editorial: Antibody validation requirements for articles published in endocrinology. Endocrinology. 2013;154:579 –580.
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Editorial: Antibody Can Get It Right: Confronting Problems of Antibody Specificity and Irreproducibility Agnes Schonbrunn, PhD Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, Houston, Texas 77225
ntibodies are indispensable research reagents. Their use in endocrinology has exploded since the original radioimmunoassays were developed to measure hormone levels. They are now routinely used in molecular endocrinology research to detect and quantitate cellular proteins and determine their distribution in tissues, in cells, and in subcellular compartments. They detect changes in protein structure produced by covalent modification during cell signaling (eg, phosphorylation), transcriptional regulation (acetylation and methylation), or protein degradation (ubiquitination). They identify and detect protein-protein and protein-DNA interactions by coimmunoprecipitation. Antibodies are used as tools to sort cells, to identify drug targets in normal and diseased tissue, and to monitor disease status and progression. And that is just the tip of the iceberg. Unfortunately, the widespread use of antibodies has also generated enormous controversy: the inability of investigators to replicate published data often results from false-positive or false-negative results produced with antibodies that have not been properly validated (1–5). The problems are particularly intense in fields that use antibodies to analyze proteins that are expressed at low levels in cells: G protein– coupled receptors, steroid hormone receptors, ion channels, transporters, and signal-transducing enzymes such as adenylyl cyclase (6 –22). In addition, ultrasensitive detection methods have compounded the problem as illustrated by studies aimed at detecting low-abundance protein–DNA interactions (eg, chromatin immunoprecipitation assays) (5, 23). So what can we do as individual scientists and as journal editors to ensure the reliability and reproducibility of the data that we generate, the data that we publish, and the data that we rely upon to formulate new hypotheses and plan future experiments?
A
doi: 10.1210/me.2014-1230
Challenge 1—Identifying a Reliable Antibody for the Job at Hand Investigators can either purchase antibodies or make their own, and there are major advantages and disadvantages to each choice. However, in either case, validation of antibodies is the responsibility of the user—validation of commercial antibodies by their suppliers is usually inadequate and frequently unreliable. The advantages of purchasing antibodies are obvious. Good antibodies can take months to produce, there are substantial up-front costs, and there is no guarantee that a particular antigen will induce antibodies of the desired properties in immunized animals. The hope is that purchasing antibodies will eliminate production delays, reduce the investment of time and money in a particular experiment, and preclude the possibility of failure to generate the desired reagent. However, identifying an appropriate antibody for purchase is no simple task: there are often dozens of antibodies available to a target protein with little information provided as to their affinity or specificity. One might think that a manufacturer’s catalog number would provide a unique identifier for a particular antibody. However, this is not the case. Vendors usually assign catalog numbers for an antibody based on the immunizing antigen, the manner in which the antibody was produced (host animal, polyclonal or monoclonal, affinity purification, etc.), and the manufacturer that produced it. In the case of polyclonal antibodies, vendors may use the same catalog number not only for different blood collections from an individual immunized animal but also for blood collections from different host animals immunized with the same antigen. Because each blood sample collected from each animal provides a unique combination of antibody clones and concentrations, this practice can result in immense lot-to-lot variability in an-
Mol Endocrinol, September 2014, 28(9):1403–1407
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Mol Endocrinol, September 2014, 28(9):1403–1407
tibodies. To add further confusion, validation data pro- the nature of the immunizing antigen is kept confidential. vided by a vendor for an antibody may not have been When investigators generate their own polyclonal antigenerated with the current lot of that antibody. Although body, they can produce both preimmune serum and extra monoclonal antibodies would not be expected to show immunizing antigen for subsequent validation experisuch lot-to-lot variability, in fact they can. For example ments. Generating monoclonal antibodies will often yield Pozner-Moulis et al (24) demonstrated that two different multiple hybridoma clones that can be compared to help lots of a monoclonal antibody to the Met tyrosine kinase ensure that any observed reactivity is specific for the tarreceptor showed opposite staining patterns in an array of get protein. Often the decision to buy an antibody or to generate more than 600 breast cancer cases: one showed nuclear and the other membranous and cytoplasmic staining. One one’s own will be determined by whether a commercial final word of caution: a particular antibody may be sold antibody has been demonstrated by others to “work” for by several distributors under different catalog numbers. a desired application and whether the antibody will be used for a short-term or a long-term project. Caveat emptor. Because the documentation provided by manufacturers is often inadequate, a number of searchable databases have been established to inventory and index antibodies Challenge 2—Validating Antibodies from multiple vendors and to list publications that have Once one or more promcited each antibody (for exising antibodies are amples, see Refs. 25–29). “It is the user’s responsibility to identified, whether purSuch databases are very helpensure that these antibodies are chased or generated inful: articles that carefully sufficiently sensitive and specific to house, it is the user’s revalidate an antibody for a produce reliable results in the sponsibility to ensure specific application often prothat these antibodies are vide the most useful guide for proposed studies. The literature sufficiently sensitive antibody selection, and au- documenting the many problems and specific to produce thors who generate such in- and incorrect conclusions resulting reliable results in the formation ought to be com- from poorly validated antibodies proposed studies. The mended and cited. However, shows that vigorous skepticism and literature documenting it is important to remember critical analysis are a must during the many problems and that the results in such articles incorrect conclusions this process (1–18).” may have been produced usresulting from poorly ing a lot that is no longer validated antibodies shows that vigorous skepticism and available. Unfortunately, many published articles do not provide catalog numbers or lot numbers for the antibod- critical analysis are a must during this process (1–18). All ies used and hence cannot be included in these databases. antibodies cross-react to some extent (1, 30). In any parBecause commercial antibodies often lack the sensitiv- ticular situation, the ratio of specific to nonspecific bindity and specificity required for an application, user vali- ing to target and off-target reactors will be determined by dation is essential. In fact, testing of purchased antibodies the relative abundance of each of the reactors in a particoften requires a substantial investment of time and ular preparation, the affinity of the antibody for each money, and there is no guarantee that a satisfactory re- reactor, the antibody concentration used in the assay, and agent will be identified for the intended use or that it will the method of sample preparation. Off-target reactivity continue to be available. When investigators choose to with polyclonal antibodies can be produced by any of the generate their own antibodies, they do so to ensure that antibody species present in the preparation. However, the work invested in antibody validation is not wasted monoclonal antibodies also show nonspecific reactivity and will result in sufficient amounts of a carefully char- (31). In fact, because mouse monoclonal antibodies tend acterized reagent to complete a study. However, there are to have lower binding affinities than rabbit polyclonal also some useful side benefits. Investigators can control antibodies and are often used at high concentrations, nonthe purity of the antigen they use for immunization, specific binding can be a prohibitive problem. A large number of thoughtful articles provide detailed whether it is a chemically synthesized peptide or a purified protein, thus helping to ensure that the resulting antibod- guidance for proper antibody validation and discuss the ies will be specific. Vendors normally do not disclose the limitation and interpretation of each type of control (13, purity of their immunogens, and, in many instances, even 30 –36). Investigators who use antibodies need to be fa-
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miliar with this literature and rigorously apply the criteria antibody produces a “clean” Western blot with no signal, recommended. It is particularly important to remember implying a lack of sensitivity for the target protein, but that the reactivity of an antibody with a target protein rather that many bands are observed on immunoblots, depends on the concentration of that protein in the prep- none of which correspond to the target protein; ie, the aration. Thus, the fact that an antibody is able to detect a antibody is both insensitive and lacks specificity. Alprotein at the high levels observed in transfected cell lines though it is understandable that antibodies produced to does not guarantee that it will do so in nontransfected native proteins may not recognize the corresponding decells or tissues. As a result, the most stringent control for natured protein after SDS-PAGE, this is not true for anantibody specificity requires comparison of antibody re- tibodies produced against short peptides. Thus, rather activity in wild-type tissues or cells to reactivity in knock- than being reassuring, a vendor’s statement that an antiout animals or cell lines in which endogenous protein peptide antibody does not work on immunoblots should expression has been silenced. For human tissue samples be cause for concern. We as well as others (24) have found in which such a knockout control is not feasible, an that immunoblotting provides a more sensitive and disalternate stringent control would be the use of multiple criminating method for antibody validation than immuantibodies recognizing different epitopes in the same nocytochemistry because it so clearly distinguishes crossreacting proteins of different molecular sizes. Moreover, target protein. Some common misconceptions have particularly im- it is far more likely that an antipeptide antibody will peded reliable antibody usage. A control frequently used “work” in Western blotting and not in immunocytoto evaluate antibody specificity is to determine the effect chemistry than the reverse. The only exception that we that preincubation with excess antigen has on antibody have encountered occurred when only a very small fraction of cells in a tissue exreactivity: the antigen pressed the target protein. In preadsorption test (31). “The most stringent control for that circumstance, the target Although this control protein constitutes such a can sometimes be use- antibody specificity requires small part of a tissue extract ful, it is certainly not comparison of antibody reactivity that it is not detectable in an sufficient. Blocking re- in wild-type tissues or cells to immunoblot. Thus, all antiactivity with excess an- reactivity in knockout animals or peptide antibodies should be tigen demonstrates that cell lines in which endogenous tested by immunoblotting tisthe reactivity is prosues and/or cells that do and duced by an antibody protein expression has been silenced. For human tissue samples do not contain the target prothat recognizes that antein to ensure (a) that the intigen. However, it does in which such a knockout control is tact protein is detected with not demonstrate that not feasible, an alternate stringent the necessary sensitivity and the antibody is specific control would be the use of (b) that reactivity with offonly for that antigen. In multiple antibodies recognizing target proteins is minimal. fact, because this condifferent epitopes in the same Although extensive validatrol is often carried out tion of many commonly used using very high concen- target protein.” trations of blocking anantibodies (eg, anti-ERK) is tigen, low-affinity as well as high-affinity antibodies in a no longer required or expected because of the enormous preparation will be blocked, and the binding of all these amount of published data that demonstrate their specificantibodies to both on-target and off-target reactors will ity and provide reliable positive controls, this is not the be inhibited. Thus, although the antigen preadsorption case for antibodies to receptors and many other proteins test can identify the population of antibodies responsible involved in cell signaling. Thus, experiments that test anfor observed reactivity, it cannot demonstrate that those tibody specificity with clear positive and negative controls antibodies are specific. are essential— otherwise an entire project may be based A false sense of security may also be provided by a statement from vendors that their antibody works for on an artifact, and valuable research resources will be immunocytochemistry or immunohistochemistry but wasted when others try to replicate published data (6 – does not work on immunoblots: such a statement pro- 16). Investigators who wish to avoid the embarrassment vides an excuse not to bother with Western blot controls. of incorrect conclusions must adopt the mantra: validate However, this statement usually does not mean that an early and often.
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Schonbrunn
Editorial
Challenge 3—Publishing Sufficient Information to Allow Critical Evaluation of Experimental Data and Facilitate Reproducibility Journals have a responsibility to help ensure that the articles they publish report valid and reproducible results. Unfortunately, journals have put increasing emphasis on reducing the length of published articles. Thus, the space devoted to describing materials and methods has been dramatically reduced over the years. The source of many reagents is missing or inadequately described. Critical control experiments have been relegated to “data not shown.” Figures display Western blots with only slivers of gels containing bands of undefined molecular weights. Frequently, only summary graphs, but no original data, are shown. These widespread practices prevent assessment of either the sensitivity or the specificity of the antibodies utilized and thus endanger the conclusions reached. The good news is that the advent of electronic publication now permits rigid space restrictions to be relaxed. Molecular Endocrinology plans to take full advantage of our change to electronic publication to ensure that the articles in our journal are reliable and reproducible. To guarantee that research reagents are clearly identified, we plan to follow the excellent example set by Endocrinology (37) and publish a full description of all antibodies used in a study. Essential information to be provided includes the following: the name of the individual or vendor who supplied the antibody, the immunizing antigen used to generate the antibody, the nature of the antibody preparation (species, polyclonal or monoclonal, and affinity purified or not), the catalog number, and, importantly, the lot number. We encourage authors to include this information in all submitted papers starting immediately and will require this information starting January 1, 2015. In addition, as a service to the research community, we encourage authors to also list antibodies that they have tested and found to be unsatisfactory. Finally, to help ensure that results in our journal are both reliable and reproducible, we commit to publishing the experimental validation for each new antibody utilized (positive and negative controls, full gels for immunoblots showing the molecular weights of all stained bands, etc.). If antibodies have been validated in a previous publication, this must be cited, and the validating experiments and results should be summarized in the text to provide confidence in the present publication. In all instances, investigators need to describe, and preferably show, the experiments demonstrating that their antibodies are specifically detecting the target protein under their
Mol Endocrinol, September 2014, 28(9):1403–1407
experimental conditions. Such supporting data may go into the body of the article or into a supplement, as the authors see fit. Antibodies are extremely powerful and sensitive reagents that provide the foundation on which many different molecular studies are built. However, they are capable of producing not just misleading but completely incorrect results. By following best practices and exercising great caution with a healthy dose of skepticism, we can ensure that published data are both reliable and reproducible and that the antibodies we use “get it right.” Agnes Schonbrunn, PhD Associate Editor
Acknowledgments I am grateful to the many colleagues who offered helpful suggestions and comments on this article. Address all correspondence and requests for reprints to: Agnes Schonbrunn, Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center at Houston, PO Box 20708, Houston, TX 77225. E-mail: [email protected]. A.S. is supported by the National Institute of Diabetes and Digestive and Kidney Diseases (Grant DK032234-26). Disclosure Summary: The author has nothing to disclose.
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