Western Blotting Analysis
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CHAPTER 35
Western Blotting Analysis Roger J. Fido, Arthur S. Tatham, and Peter R. Shewry
1. Introduction The immunodetection of proteins bound to a membrane has widespread applications in plant biochemistry and molecular biology, including the identification and semiquantitative determination of foreign proteins expressed in transgenic plants. The approach is usually applied to protein transferred from electrophoretic separations (see Chapter 34), which allows positive identification to be combined with the provision of information about the protein M^ charge, pi, and so on. This is commonly called Western blotting, and provides the operator with a wide range of options for choice of membrane type, transfer system, and detection system. We will initially discuss these options, and then provide detailed step-by-step instructions for a well-established method of protein transfer and identification using an enzyme-labeled second antibody. In addition, we will discuss two other applications of protein blotting that provide rapid but less precise results. These are analysis of tissue extracts using dot blotting, and "squash" blots of whole plants or plant organs. These different applications of protein blotting and immunochemical detection facilitate the rapid screening of multiple samples for protein expression, and detailed analysis of the amount and properties of proteins expressed in individual plants or plant organs. An example of the use of Western blotting to study the expression and processing of a protein encoded by a transgene is shown by Higgins and Spencer (1), (see also Fig. 1).
Callus Stem Root Leaf
Seed
Fig, I. The distribution of polypeptides related to pea vicilin in transgenic tobacco plants (14/2) transformed with a 35S-vicilin chimeric gene. (A) In whole tissue extracts of callus, stem, root, leaf, and seed; and (B) In immunopurified fractions from leaves of various ages. The vicilin related polypeptides were detected after fractionation by SDS-PAGE, blotted onto nitrocellulose and detected by alkaline phosphatase-coupled antibodies (from Higgins and Spencer, 1991 [1]).
Western Blotting Analysis
425
1.2. Transfer Membranes Several types of membrane are available for blotting, the choice depending on the protein under study. The most commonly used is nitrocellulose (NC), which is available either pure or supported, and in a range of formats and pore sizes. Although a 0.45-|Lim pore size is widely used, low molecular weight proteins (i.e., below 20 kDa) require a smaller pore size, with 0.2 (j.m being recommended. Suppliers such as Schleicher and Schuell (Keene, NH) provide pure NC membrane in six pore sizes. Because pure nitrocellulose membrane is fragile and will tear and crack easily, it must be handled carefully, and always with gloved hands. Although more suitable for nucleic acid techniques, nylon membranes are an alternative to NC and are available in charged (e.g., Zeta-Probe from Bio-Rad, Richmond, CA) or uncharged (e.g., Hybond-N from Amersham International, Arlington Heights, IL) forms. They are resistant to tearing, very pliable, and have advantages over NC in certain circumstances, including a higher protein-binding capacity. However, there are also some limitations to their use. Because of their increased binding capacity they can give high nonspecific binding and consequently require increased concentrations of blocking agent. In addition, nylon membranes are incompatible with the most commonly used anionic protein stains, such as Amido Black and Coomassie blue. The polyvinyldifluoride (PVDF) membranes, such as Immobilon (Millipore Corp., Bedford, MA) and ProBlott (Applied Biosystems Inc., Foster City, CA) can be used for automated microsequencing of proteins directly after transfer. They can be used in the same way as NC after initially prewetting in 100% methanol for a short time (see Immobiline Technical Bulletin, Millipore). In a recent comparison of different membrane types it was shown that the PVDF-based membranes were the most efficient for microsequencing analysis (5). Many companies, including Bio-Rad (Poole, UK) and BDH, provide free guides to blotting membranes. 2. Materials 2.1. Electroblotting of Gels 1. Transfer buffer: 25 mMTris, 192 mMglycine, and 20% methanol, pH 8.3, with or without 0.02% (w/v) SDS. 2. Nitrocellulose transfer membrane cut to size of gel. 3. Filter papers (Whatman [Maidstone, UK] 3MM) cut to the size of the gel.
Western Blotting Analysis Roger J. Fido, Arthur S. Tatham, and Peter R. Shewry
1. Introduction The immunodetection of proteins bound to a membrane has widespread applications in plant biochemistry and molecular biology, including the identification and semiquantitative determination of foreign proteins expressed in transgenic plants. The approach is usually applied to protein transferred from electrophoretic separations (see Chapter 34), which allows positive identification to be combined with the provision of information about the protein M^ charge, pi, and so on. This is commonly called Western blotting, and provides the operator with a wide range of options for choice of membrane type, transfer system, and detection system. We will initially discuss these options, and then provide detailed step-by-step instructions for a well-established method of protein transfer and identification using an enzyme-labeled second antibody. In addition, we will discuss two other applications of protein blotting that provide rapid but less precise results. These are analysis of tissue extracts using dot blotting, and "squash" blots of whole plants or plant organs. These different applications of protein blotting and immunochemical detection facilitate the rapid screening of multiple samples for protein expression, and detailed analysis of the amount and properties of proteins expressed in individual plants or plant organs. An example of the use of Western blotting to study the expression and processing of a protein encoded by a transgene is shown by Higgins and Spencer (1), (see also Fig. 1).
Callus Stem Root Leaf
Seed
Fig, I. The distribution of polypeptides related to pea vicilin in transgenic tobacco plants (14/2) transformed with a 35S-vicilin chimeric gene. (A) In whole tissue extracts of callus, stem, root, leaf, and seed; and (B) In immunopurified fractions from leaves of various ages. The vicilin related polypeptides were detected after fractionation by SDS-PAGE, blotted onto nitrocellulose and detected by alkaline phosphatase-coupled antibodies (from Higgins and Spencer, 1991 [1]).
Western Blotting Analysis
425
1.2. Transfer Membranes Several types of membrane are available for blotting, the choice depending on the protein under study. The most commonly used is nitrocellulose (NC), which is available either pure or supported, and in a range of formats and pore sizes. Although a 0.45-|Lim pore size is widely used, low molecular weight proteins (i.e., below 20 kDa) require a smaller pore size, with 0.2 (j.m being recommended. Suppliers such as Schleicher and Schuell (Keene, NH) provide pure NC membrane in six pore sizes. Because pure nitrocellulose membrane is fragile and will tear and crack easily, it must be handled carefully, and always with gloved hands. Although more suitable for nucleic acid techniques, nylon membranes are an alternative to NC and are available in charged (e.g., Zeta-Probe from Bio-Rad, Richmond, CA) or uncharged (e.g., Hybond-N from Amersham International, Arlington Heights, IL) forms. They are resistant to tearing, very pliable, and have advantages over NC in certain circumstances, including a higher protein-binding capacity. However, there are also some limitations to their use. Because of their increased binding capacity they can give high nonspecific binding and consequently require increased concentrations of blocking agent. In addition, nylon membranes are incompatible with the most commonly used anionic protein stains, such as Amido Black and Coomassie blue. The polyvinyldifluoride (PVDF) membranes, such as Immobilon (Millipore Corp., Bedford, MA) and ProBlott (Applied Biosystems Inc., Foster City, CA) can be used for automated microsequencing of proteins directly after transfer. They can be used in the same way as NC after initially prewetting in 100% methanol for a short time (see Immobiline Technical Bulletin, Millipore). In a recent comparison of different membrane types it was shown that the PVDF-based membranes were the most efficient for microsequencing analysis (5). Many companies, including Bio-Rad (Poole, UK) and BDH, provide free guides to blotting membranes. 2. Materials 2.1. Electroblotting of Gels 1. Transfer buffer: 25 mMTris, 192 mMglycine, and 20% methanol, pH 8.3, with or without 0.02% (w/v) SDS. 2. Nitrocellulose transfer membrane cut to size of gel. 3. Filter papers (Whatman [Maidstone, UK] 3MM) cut to the size of the gel.
