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Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology
J. Biochem. Biophys. Methods 70 (2007) 679 – 682 www.elsevier.com/locate/jbbm
Short note
Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology Toshiyuki Hata a , Manabu Nakayama a,b,⁎ a
Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, 2–6-7, Kazusa-Kamatari, Kisarazu, Chiba 292–0818 Japan b Department of Human Genome Technology, Kazusa DNA Research Institute, 2–6-7, Kazusa-Kamatari, Kisarazu, Chiba 292–0818 Japan Received 15 September 2006; received in revised form 22 January 2007; accepted 26 January 2007
Abstract Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies. © 2007 Elsevier B.V. All rights reserved. Keywords: HaloTag; HaloLink; Immunoaffinity; Haloalkane dehalogenase; KIAA1440; Functional genomics
Specific antibodies against a protein still prove to be powerful tools to study the function of protein. When antibodies are needed against a cloned gene product, one way of obtaining a superior source of antigen required for developing antibodies is to overexpress either the full-length protein or various fusion proteins (tagged coding regions) in bacteria. To produce polyclonal antibodies, rabbits are then immunized with the purified, tagged antigen. Although this procedure is now routine, it does not always produce pure, high affinity, antigen-specific polyclonal antibodies. When the serum is tested by immunoblotting, cross-reactive bands are sometimes detected in addition to the protein of interest. Polyclonal antibodies are purified from the immune serum by immunoaffinity purification, the only method currently available to purify antigen-specific antibodies from a polyclonal pool of antibodies [1]. In immunoaffinity purification, the ⁎ Corresponding author. Manabu Nakayama, 2–6-7 Kazusa-Kamatari, Kisarazu, Chiba 292–0818 Japan. Tel.: +81 438 52 3909; fax: +81 438 52 3946. E-mail address: [email protected] (M. Nakayama). 0165-022X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2007.01.014
antigen is bound covalently to a solid support, such as a resin bead; the immune serum is mixed with the beads to allow antigen-specific antibodies to bind the antigen-coupled beads; and unbound, non-specific antibodies are removed by washing. The specific antibodies are then eluted. The major advantage of this method is its unique ability to isolate specific antibodies from a mixed pool. The major disadvantages of this method are that it (1) requires large amounts of pure antigen; and (2) requires the covalent linking of pure antigen to the bead support, which involves complicated steps (e.g., HiTrap NHSactivated columns). Here, we describe a simple procedure for the small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology, a technology that uses HaloTag Protein and a system of interchangeable synthetic ligands that specifically and covalently bind to the HaloTag Protein. These ligands impart multiple functions to a HaloTag fusion protein, including the ability to image subcellular proteins and protein immobilization. The HaloLink Resin enables covalent and oriented attachment of HaloTag fusion proteins to the surface of Sepharose via the HaloTag Ligand.
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T. Hata, M. Nakayama / J. Biochem. Biophys. Methods 70 (2007) 679–682
Fig. 1A shows a simple schematic of the immunoaffinity purification of polyclonal antibodies. With this standard immunoaffinity purification procedure, specific antibodies are
obtained at the end of three steps: (1) purification of the antigen (1–3 mg), (2) coupling of the antigen to a HiTrap NHSactivated column, and (3) affinity column purification of the
T. Hata, M. Nakayama / J. Biochem. Biophys. Methods 70 (2007) 679–682
polyclonal serum (5–10 ml). Although a sufficient amount of specific antibodies is obtained, this method is tedious and timeconsuming. With our new rapid HaloTag-based procedure, however, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the by HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Fig. 1C shows specificity analysis results of unpurified polyclonal antibodies serum. Immunoblots of mouse brain extracts treated with different dilutions of polyclonal serum obtained by immunizing a rabbit with an His-tagged fusion protein (amino acids 241–544 of mouse KIAA1440 protein) produced discrete immunoreactive bands of the expected molecular weight for KIAA1440 [2] (Fig. 1C and data not shown). Numerous minor cross-reactive bands, however, were also produced. To determine whether HaloTag technology can be applied to immunoaffinity purification of polyclonal antibodies to KIAA1440, we fused maltose binding protein (MBP)-conjugated HaloTag to amino acids 241–386 of mouse KIAA1440 protein, expressed this fusion protein in E. coli, and used it as antigen of immunoaffinity purification procedure. We also expressed MBP-conjugated HaloTag separately, to be used as a control. After incubating E. coli extract (100 μl) containing soluble fusion protein with 50 μl of HaloLink Resin (50% suspension) for 1 h at room temperature, we measured unbound MBP-HaloTag and MBP-HaloTag-KIAA1440 levels. Although we found decreased levels of unbound MBP-HaloTag, most likely due to binding to resin rather than to unrelated E. coli proteins, we did not find a similar significant decrease in unbound MBP-HaloTag-KIAA1440, suggesting that the KIAA1440 fragment may have sterically hindered MBPHaloTag from binding to HaloTag Resin, but only a small amount of MBP-HaloTag-KIAA1440 can bind to HaloLink Resin. Therefore, to reduce steric hindrance and to maintain strong activity of HaloTag Protein, we should insert a polypeptide linker composed of [Ser–Gly4] repeats between the
681
HaloTag and the KIAA1440 fragment, as suggested by the manufacturer. After several washes, we incubated HaloLink Resin-coupled MBP-HaloTag-KIAA1440 with 100 μl of diluted immune serum (1:10) for 1 h at 4 °C, washed three times with high salt buffer (500 mM NaCl, 10 mM Tris–HCl [pH 7.5]) to remove unbound, non-specific antibodies, then eluted bound, KIAA1440-specific antibodies with acidic glycine buffer (100 mM glycine [pH 2.5]). The same procedure was also carried out with HaloLink Resin-coupled MBP-HaloTag. We then analyzed the eluted antibodies by SDS-PAGE (Fig. 1D). We detected polyclonal antibodies in elutes from HaloLinked Resincoupled MBP-HaloTag-KIAA1440 but not in those from HaloLinked Resin-coupled MBP-HaloTag (Fig. 1D; lane 6 cf. lane 5). Immunoblots of mouse brain extracts treated with the eluates confirmed that KIAA1440-specific antibodies were successfully purified by the HaloLinked Resin-coupled MBPHaloTag-KIAA1440 (Fig. 1C; lane 2). Compared with other protein tag systems, a unique feature of the HaloLink Resin system is that proteins of interest bind covalently to the resin, thereby allowing researchers to thoroughly wash away non-specifically bound proteins while retaining the resin-bound fusion protein of interest. This feature allowed us to use a fairly strong acid buffer—100 mM Glycine (pH 2.5) or 4 M urea—to vigorously pre-wash the resin immediately before the immune serum was passed through the column for antibody-to-antigen/resin binding, or to wash the used resin for repeatable purification (regeneration of resin-coupled antigen). The HaloTag Protein is an engineered monomer haloalkane dehalogenase from Rhodococcus rhodochrous. This dehalogenase contains a His272Phe substitution that facilitates covalent binding to a protein of interest. Another tag capable of covalent binding is SNAP-tag, an engineered mutant of human alkylguanine-DNA alkyl transferase (AGT), a DNA repair protein. When SNAP-tag is used for antibody purification in place of HaloTag, one should keep in mind that antibodies that bind with SNAP-tag can produce non-specific staining, especially when used for staining immunoblots of human or mouse extracts. Immunostaining such blots with SNAP-tag-purified antibodies might produce an AGT noise band, potentially.
Fig. 1. Overview of a rapid, single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag Technology. (A) Comparison between a standard immunoaffinity purification procedure and our rapid HaloTag procedure. (B) Schematic diagram showing the importance of surveying suitable antigenic determinants or epitopes for use in immunoaffinity purification to obtain high affinity, high specificity antibodies against a protein of interest from a polyclonal pool of antibodies. (C) Specificity analysis of polyclonal antibodies affinity-purified using a HaloLink Resin. Mouse whole brain extracts (100 μg) were separated by 2–15% gradient SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes. Immunoblot of mouse whole brain extracts immunostained with polyclonal anti-mKIAA1440 serum (lane 1) or eluate from HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 (lane 2). Immunoreactive complexes were detected by treating the membranes with HRP-conjugated anti-rabbit IgG followed by the ECL plus detection system. The closed triangle points to the expected molecular weight of fulllength mouse KIAA1440 protein. Open triangles point to some typical cross-reactive bands. (D) SDS-PAGE of eluates (10 μl each) from immunoaffinity resins containing either HaloLinked Resin-coupled MBP-HaloTag or HaloLinked Resin-coupled MBP-HaloTag-KIAA1440. After 10% SDS-PAGE, gels were stained with CBB. Lane 1, MBP-HaloTag (soluble fraction) overexpressed in E. coli; lane 2, MBP-HaloTag-KIAA1440 (soluble fraction) overexpressed in E. coli; lane 3, unbound fraction from the HaloLinked Resin-coupled MBP-HaloTag column after the HaloLink Resin reaction; lane 4, unbound fraction from the HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 column after the HaloLink Resin reaction; lane 5, bound fraction eluted with 100 mM glycine (pH 2.5) after immunoaffinity purification using the HaloLinked Resin-coupled MBP-HaloTag column; lane 6, bound fraction eluted with 100 mM glycine (pH 2.5) after immunoaffinity purification using the HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 column. Open and filled arrowheads point to MBP-HaloTag and MBP-HaloTag-KIAA1440, respectively. Arrow points to the heavy chains of antibodies affinity-purified by our rapid single-tube method (lane 6). (E) Aliquots (1 μl each) of the glycine buffer-eluted antibodies, themselves, from either HaloLinked Resin-coupled MBP-HaloTag (lane 1) or HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 (lane 2) were also detected by incubating the membranes with HRP-conjugated anti-rabbit IgG followed by ECL Plus detection system. Arrow points to the heavy chains of antibodies affinity-purified by our rapid single-tube method (lane 2).
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T. Hata, M. Nakayama / J. Biochem. Biophys. Methods 70 (2007) 679–682
A weakness of our procedure is that it produces only a small amount of purified antibodies, which is a consequence of performing simultaneously the purification and covalent coupling steps in a single tube. Of course, it would be possible to scale up our method if we increased the volume of HaloLink Resin. We strongly recommend, however, that those desiring to use the HaloTag method should use our single-tube method as an initial pilot test before attempting large-scale immunoaffinity purification of antibodies. Fig. 1B outlines the principle underlying why the appropriate antigenic determinant or epitope must be used for immunoaffinity purification in order to obtain high affinity, high specificity antibodies from a polyclonal pool of antibodies. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple,
potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies. Acknowledgements We are grateful to E. Suzuki and S. Minorikawa for their excellent technical assistance. We also thank Dr. O. Ohara for his critical reading of this manuscript and for valuable discussions. This study was supported by grants from the Kazusa DNA Research Institute and in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. References [1] Harlow E, Lane D. Antibodies: A Laboratory manual.. New York: Cold Spring Harbor Laboratory Press; 1988. [2] Nakayama M, Iida M, Koseki H, Ohara O. A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins. FASEB J 2006;20:1718–20.
Short note
Rapid single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology Toshiyuki Hata a , Manabu Nakayama a,b,⁎ a
Laboratory of Pharmacogenomics, Graduate School of Pharmaceutical Sciences, Chiba University, 2–6-7, Kazusa-Kamatari, Kisarazu, Chiba 292–0818 Japan b Department of Human Genome Technology, Kazusa DNA Research Institute, 2–6-7, Kazusa-Kamatari, Kisarazu, Chiba 292–0818 Japan Received 15 September 2006; received in revised form 22 January 2007; accepted 26 January 2007
Abstract Even in this era of advanced biotechniques, specific antibodies against a protein still prove to be powerful tools to study proteins and their functions. The polyclonal antisera obtained from the immunized rabbits, however, are not always pure, high affinity, antigen-specific polyclonal antibodies. With our new rapid HaloTag-based procedure, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple, potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies. © 2007 Elsevier B.V. All rights reserved. Keywords: HaloTag; HaloLink; Immunoaffinity; Haloalkane dehalogenase; KIAA1440; Functional genomics
Specific antibodies against a protein still prove to be powerful tools to study the function of protein. When antibodies are needed against a cloned gene product, one way of obtaining a superior source of antigen required for developing antibodies is to overexpress either the full-length protein or various fusion proteins (tagged coding regions) in bacteria. To produce polyclonal antibodies, rabbits are then immunized with the purified, tagged antigen. Although this procedure is now routine, it does not always produce pure, high affinity, antigen-specific polyclonal antibodies. When the serum is tested by immunoblotting, cross-reactive bands are sometimes detected in addition to the protein of interest. Polyclonal antibodies are purified from the immune serum by immunoaffinity purification, the only method currently available to purify antigen-specific antibodies from a polyclonal pool of antibodies [1]. In immunoaffinity purification, the ⁎ Corresponding author. Manabu Nakayama, 2–6-7 Kazusa-Kamatari, Kisarazu, Chiba 292–0818 Japan. Tel.: +81 438 52 3909; fax: +81 438 52 3946. E-mail address: [email protected] (M. Nakayama). 0165-022X/$ - see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jbbm.2007.01.014
antigen is bound covalently to a solid support, such as a resin bead; the immune serum is mixed with the beads to allow antigen-specific antibodies to bind the antigen-coupled beads; and unbound, non-specific antibodies are removed by washing. The specific antibodies are then eluted. The major advantage of this method is its unique ability to isolate specific antibodies from a mixed pool. The major disadvantages of this method are that it (1) requires large amounts of pure antigen; and (2) requires the covalent linking of pure antigen to the bead support, which involves complicated steps (e.g., HiTrap NHSactivated columns). Here, we describe a simple procedure for the small-scale affinity purification of polyclonal antibodies using HaloTag™ Technology, a technology that uses HaloTag Protein and a system of interchangeable synthetic ligands that specifically and covalently bind to the HaloTag Protein. These ligands impart multiple functions to a HaloTag fusion protein, including the ability to image subcellular proteins and protein immobilization. The HaloLink Resin enables covalent and oriented attachment of HaloTag fusion proteins to the surface of Sepharose via the HaloTag Ligand.
680
T. Hata, M. Nakayama / J. Biochem. Biophys. Methods 70 (2007) 679–682
Fig. 1A shows a simple schematic of the immunoaffinity purification of polyclonal antibodies. With this standard immunoaffinity purification procedure, specific antibodies are
obtained at the end of three steps: (1) purification of the antigen (1–3 mg), (2) coupling of the antigen to a HiTrap NHSactivated column, and (3) affinity column purification of the
T. Hata, M. Nakayama / J. Biochem. Biophys. Methods 70 (2007) 679–682
polyclonal serum (5–10 ml). Although a sufficient amount of specific antibodies is obtained, this method is tedious and timeconsuming. With our new rapid HaloTag-based procedure, however, specific antibodies are obtained in just two, short steps: (1) simultaneous purification and covalent coupling of the antigen to Sepharose resin via the by HaloTag and HaloLink reaction, and (2) affinity column purification of the polyclonal serum (10 μl). The combined antigen purification and coupling step requires only 1 h of room-temperature incubation, plus successive washing steps. Fig. 1C shows specificity analysis results of unpurified polyclonal antibodies serum. Immunoblots of mouse brain extracts treated with different dilutions of polyclonal serum obtained by immunizing a rabbit with an His-tagged fusion protein (amino acids 241–544 of mouse KIAA1440 protein) produced discrete immunoreactive bands of the expected molecular weight for KIAA1440 [2] (Fig. 1C and data not shown). Numerous minor cross-reactive bands, however, were also produced. To determine whether HaloTag technology can be applied to immunoaffinity purification of polyclonal antibodies to KIAA1440, we fused maltose binding protein (MBP)-conjugated HaloTag to amino acids 241–386 of mouse KIAA1440 protein, expressed this fusion protein in E. coli, and used it as antigen of immunoaffinity purification procedure. We also expressed MBP-conjugated HaloTag separately, to be used as a control. After incubating E. coli extract (100 μl) containing soluble fusion protein with 50 μl of HaloLink Resin (50% suspension) for 1 h at room temperature, we measured unbound MBP-HaloTag and MBP-HaloTag-KIAA1440 levels. Although we found decreased levels of unbound MBP-HaloTag, most likely due to binding to resin rather than to unrelated E. coli proteins, we did not find a similar significant decrease in unbound MBP-HaloTag-KIAA1440, suggesting that the KIAA1440 fragment may have sterically hindered MBPHaloTag from binding to HaloTag Resin, but only a small amount of MBP-HaloTag-KIAA1440 can bind to HaloLink Resin. Therefore, to reduce steric hindrance and to maintain strong activity of HaloTag Protein, we should insert a polypeptide linker composed of [Ser–Gly4] repeats between the
681
HaloTag and the KIAA1440 fragment, as suggested by the manufacturer. After several washes, we incubated HaloLink Resin-coupled MBP-HaloTag-KIAA1440 with 100 μl of diluted immune serum (1:10) for 1 h at 4 °C, washed three times with high salt buffer (500 mM NaCl, 10 mM Tris–HCl [pH 7.5]) to remove unbound, non-specific antibodies, then eluted bound, KIAA1440-specific antibodies with acidic glycine buffer (100 mM glycine [pH 2.5]). The same procedure was also carried out with HaloLink Resin-coupled MBP-HaloTag. We then analyzed the eluted antibodies by SDS-PAGE (Fig. 1D). We detected polyclonal antibodies in elutes from HaloLinked Resincoupled MBP-HaloTag-KIAA1440 but not in those from HaloLinked Resin-coupled MBP-HaloTag (Fig. 1D; lane 6 cf. lane 5). Immunoblots of mouse brain extracts treated with the eluates confirmed that KIAA1440-specific antibodies were successfully purified by the HaloLinked Resin-coupled MBPHaloTag-KIAA1440 (Fig. 1C; lane 2). Compared with other protein tag systems, a unique feature of the HaloLink Resin system is that proteins of interest bind covalently to the resin, thereby allowing researchers to thoroughly wash away non-specifically bound proteins while retaining the resin-bound fusion protein of interest. This feature allowed us to use a fairly strong acid buffer—100 mM Glycine (pH 2.5) or 4 M urea—to vigorously pre-wash the resin immediately before the immune serum was passed through the column for antibody-to-antigen/resin binding, or to wash the used resin for repeatable purification (regeneration of resin-coupled antigen). The HaloTag Protein is an engineered monomer haloalkane dehalogenase from Rhodococcus rhodochrous. This dehalogenase contains a His272Phe substitution that facilitates covalent binding to a protein of interest. Another tag capable of covalent binding is SNAP-tag, an engineered mutant of human alkylguanine-DNA alkyl transferase (AGT), a DNA repair protein. When SNAP-tag is used for antibody purification in place of HaloTag, one should keep in mind that antibodies that bind with SNAP-tag can produce non-specific staining, especially when used for staining immunoblots of human or mouse extracts. Immunostaining such blots with SNAP-tag-purified antibodies might produce an AGT noise band, potentially.
Fig. 1. Overview of a rapid, single-tube method for small-scale affinity purification of polyclonal antibodies using HaloTag Technology. (A) Comparison between a standard immunoaffinity purification procedure and our rapid HaloTag procedure. (B) Schematic diagram showing the importance of surveying suitable antigenic determinants or epitopes for use in immunoaffinity purification to obtain high affinity, high specificity antibodies against a protein of interest from a polyclonal pool of antibodies. (C) Specificity analysis of polyclonal antibodies affinity-purified using a HaloLink Resin. Mouse whole brain extracts (100 μg) were separated by 2–15% gradient SDS-PAGE. Separated proteins were transferred onto nitrocellulose membranes. Immunoblot of mouse whole brain extracts immunostained with polyclonal anti-mKIAA1440 serum (lane 1) or eluate from HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 (lane 2). Immunoreactive complexes were detected by treating the membranes with HRP-conjugated anti-rabbit IgG followed by the ECL plus detection system. The closed triangle points to the expected molecular weight of fulllength mouse KIAA1440 protein. Open triangles point to some typical cross-reactive bands. (D) SDS-PAGE of eluates (10 μl each) from immunoaffinity resins containing either HaloLinked Resin-coupled MBP-HaloTag or HaloLinked Resin-coupled MBP-HaloTag-KIAA1440. After 10% SDS-PAGE, gels were stained with CBB. Lane 1, MBP-HaloTag (soluble fraction) overexpressed in E. coli; lane 2, MBP-HaloTag-KIAA1440 (soluble fraction) overexpressed in E. coli; lane 3, unbound fraction from the HaloLinked Resin-coupled MBP-HaloTag column after the HaloLink Resin reaction; lane 4, unbound fraction from the HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 column after the HaloLink Resin reaction; lane 5, bound fraction eluted with 100 mM glycine (pH 2.5) after immunoaffinity purification using the HaloLinked Resin-coupled MBP-HaloTag column; lane 6, bound fraction eluted with 100 mM glycine (pH 2.5) after immunoaffinity purification using the HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 column. Open and filled arrowheads point to MBP-HaloTag and MBP-HaloTag-KIAA1440, respectively. Arrow points to the heavy chains of antibodies affinity-purified by our rapid single-tube method (lane 6). (E) Aliquots (1 μl each) of the glycine buffer-eluted antibodies, themselves, from either HaloLinked Resin-coupled MBP-HaloTag (lane 1) or HaloLinked Resin-coupled MBP-HaloTag-KIAA1440 (lane 2) were also detected by incubating the membranes with HRP-conjugated anti-rabbit IgG followed by ECL Plus detection system. Arrow points to the heavy chains of antibodies affinity-purified by our rapid single-tube method (lane 2).
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T. Hata, M. Nakayama / J. Biochem. Biophys. Methods 70 (2007) 679–682
A weakness of our procedure is that it produces only a small amount of purified antibodies, which is a consequence of performing simultaneously the purification and covalent coupling steps in a single tube. Of course, it would be possible to scale up our method if we increased the volume of HaloLink Resin. We strongly recommend, however, that those desiring to use the HaloTag method should use our single-tube method as an initial pilot test before attempting large-scale immunoaffinity purification of antibodies. Fig. 1B outlines the principle underlying why the appropriate antigenic determinant or epitope must be used for immunoaffinity purification in order to obtain high affinity, high specificity antibodies from a polyclonal pool of antibodies. Because different regions of an antigen can elicit the production of low affinity antibodies with relatively high cross-reactivity, the best way to produce high affinity antibodies against a protein of interest is to survey all antigenic determinants of that protein and identify the epitopes that result in the production of antibodies with a high affinity and specificity for that protein. Because our HaloTag procedure is quite rapid and simple,
potential epitopes can be assessed with relatively little effort for their ability to elicit the production of highly specific antibodies. Acknowledgements We are grateful to E. Suzuki and S. Minorikawa for their excellent technical assistance. We also thank Dr. O. Ohara for his critical reading of this manuscript and for valuable discussions. This study was supported by grants from the Kazusa DNA Research Institute and in part by Grants-in-Aid from the Ministry of Education, Culture, Sports, Science and Technology, the Japanese Government. References [1] Harlow E, Lane D. Antibodies: A Laboratory manual.. New York: Cold Spring Harbor Laboratory Press; 1988. [2] Nakayama M, Iida M, Koseki H, Ohara O. A gene-targeting approach for functional characterization of KIAA genes encoding extremely large proteins. FASEB J 2006;20:1718–20.