Current Protocols in Food Analytical Chemistry

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Current Protocols in Food Analytical Chemistry

FOREWORD

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ccurate, precise, sensitive, and rapid analytical determinations are as essential in food science and technology as in chemistry, biochemistry, and other physical and biological sciences. In many cases, the same methodologies are used. How does one, especially a young scientist, select the best methods to use? A review of original publications in a given field indicates that some methods are cited repeatedly by many noted researchers and analysts, but with some modifications adapting them to the specific material analyzed. Official analytical methods have been adopted by some professional societies, such as the Official Methods of Analysis (Association of Official Analytical Chemists), Official Methods and Recommendation Practices (American Oil Chemists’ Society), and Official Methods of Analysis (American Association of Cereal Chemists).

The objective of Current Protocols in Food Analytical Chemistry is to provide the type of detailed instructions and comments that an expert would pass on to a competent technician or graduate student who needs to learn and use an unfamiliar analytical procedure, but one that is routine in the lab of an expert or in the field. What factors can be used to predetermine the quality and utility of a method? An analyst must consider the following questions: Do I need a proximate analytical method that will determine all the protein, or carbohydrate, or lipid, or nucleic acid in a biological material? Or do I need to determine one specific chemical compound among the thousands of compounds found in a food? Do I need to determine one or more physical properties of a food? How do I obtain a representative sample? What size sample should I collect? How do I store my samples until analysis? What is the precision (reproducibility) and accuracy of the method or what other compounds and conditions could interfere with the analysis? How do I determine whether the results are correct, as well as the precision and accuracy of a method? How do I know that my standard curves are correct? What blanks, controls and internal standards must be used? How do I convert instrumental values (such as absorbance) to molar concentrations? How many times should I repeat the analysis? And how do I report my results with appropriate standard deviation and to the correct number of significant digits? Is a rate of change method (i.e., velocity as in enzymatic assays) or a static method (independent of time) needed? Current Protocols in Food Analytical Chemistry will provide answers to these questions. Analytical instrumentation has evolved very rapidly during the last 20 years as physicists, chemists, and engineers have invented highly sensitive spectrophotometers, polarometers, balances, etc. Chemical analyses can now be made using milligram, microgram, nanogram, or picogram amounts of materials within a few minutes, rather than previously when grams or kilograms of materials were required by multistep methods requiring hours or days of preparation and analysis. Current Protocols in Food Analytical Chemistry provides state-of-the-art methods to take advantage of the major advances in sensitivity, precision, and accuracy of current instrumentation. How do chemical analyses of foods differ from analyses used in chemistry, biochemistry and biology? The same methods and techniques are often used; only the purpose of the analysis may differ. But foods are to be used by people. Therefore, methodology to determine safety (presence of dangerous microbes, pesticides, and toxicants), acceptability (flavor, odor, color, texture), and nutritional quality (essential vitamins, minerals, amino acids, and lipids) are essential analyses. Current Protocols in Food Analytical Chemistry is designed to meet all these requirements. John Whitaker Davis, California Contributed by John Whitaker Current Protocols in Food Analytical Chemistry (2001) Copyright © 2001 by John Wiley & Sons, Inc.

Current Protocols in Food Analytical Chemistry

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PREFACE

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ccurate and state-of-the-art analysis of food composition is of interest and concern to a divergent clientele including research workers in academic, government, and industrial settings, regulatory scientists, analysts in private commercial laboratories, and quality control professionals in small and large companies. Some methods are empirical, some commodity specific, and many have been widely accepted as standard methods for years. Others are at the cutting edge of new analytical methodology and are rapidly changing. A common denominator within this diverse group of methods is the desire for detailed descriptions of how to carry out analytical procedures. A frustration of many authors and readers of peer-reviewed journals is the brevity of most Materials and Methods sections. There is editorial pressure to minimize description of experimental details and eliminate advisory comments. When one needs to undertake an analytical procedure with which one is unfamiliar, it is prudent to communicate first-hand with one experienced with the methodology. This may require a personal visit to another laboratory and/or electronic or phone communication with someone who has expertise in the procedure. An objective of Current Protocols in Food Analytical Chemistry is to provide exactly this kind of detailed information which personal contact would provide. Authors are instructed to present the kind of details and advisory comments they would give to a graduate student or technician who has competent laboratory skills and who has come to them to learn how to carry out an analytical procedure for which the author has expertise. Some basic food analytical methods such as determination of Brix, pH, titratable acidity, total proteins, and total lipids are basic to food analysis and grounded in procedures which have had wide-spread acceptance for a long time. Such methods cannot be ignored and are included in this manual. Others, such as analysis of cell-wall polysaccharides (Chapter E3), analysis of aroma volatiles (Chapter G1), and compressive measurement of solids and semisolids (Chapter H2), require use of advanced chemical and physical methods and sophisticated instrumentation. These methods are particularly prone to rapid change and evolution. Current Protocols capitalizes on today’s electronic communication technologies and provides a mechanism for updates, additions, and revisions. The publication is available in loose-leaf binder, CD-ROM, or Online formats. Supplements are published quarterly. Users have the opportunity to provide feedback so that individual units can be clarified, modified, and expanded. Thus Current Protocols in Food Analytical Chemistry should be viewed as a dynamic resource which will be constantly up-dated. In organizing Currrent Protocols in Food Analytical Chemistry we chose to categorize on a disciplinary rather than a commodity basis. Included are chapters on water, proteins, enzymes, lipids, carbohydrates, colors, flavors, and textural components. We have made an effort to select methods which are applicable to all commodities. However, it is impossible to address the unique and special criteria required for analysis of all commodities and all processed forms. There are several professional and trade organizations which focus on their specific commodities, e.g., cereals, wines, lipids, fisheries, and meats. Their methods manuals and professional journals should be consulted, particularly for specialized, commodity-specific analyses. With respect to our Editorial Board, we have selected scientists who are widely regarded as being authorities in their field. Their research productivity is impressive, and they are all professors who are experienced in training undergraduate and graduate students, technicians, post-doctoral students, and visiting scientists in analytical procedures. This common experience provides insight regarding the types of experimental details needed to clarify how procedures are to be conducted.

Current Protocols in Food Analytical Chemistry

Contributed by Ronald E. Wrolstad, Terry E. Acree, Haejung An, Eric A. Decker, Michael H. Penner, David S. Reid, Steven J. Schwartz, Charles F. Shoemaker, Denise M. Smith, and Peter Sporns

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Current Protocols in Food Analytical Chemistry (2003) iii-vi Copyright © 2003 by John Wiley & Sons, Inc.

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HOW TO USE THIS MANUAL Format and Organization This publication is available in looseleaf, CD-ROM, and Online formats. For loose-leaf purchasers, a binder is provided to accommodate the growth of the manual via the quarterly update service. The looseleaf format of the binder allows easy insertion of new pages, units, and chapters that are added. The index and table of contents are updated with each supplement. Purchasers of the CD-ROM and Intranet versions receive a completely new disc every quarter and should dispose of their outdated discs. The material covered in all versions is identical. Subjects in this manual are organized by sections and chapters, and protocols are contained in units. Units generally describe a method and include one or more protocols with listings of materials, steps and annotations, recipes for unique reagents and solutions, and commentaries on the “hows” and “whys” of the method; there are also “overview” units containing theoretical discussions that lay the foundation for subsequent protocols. Page numbering in the looseleaf version reflects the modular arrangement by unit; for example, page D2.1.1 refers to Section D (Lipids), Chapter D2 (Lipid Oxidation/Stability), UNIT D2.1 (Measurement of Primary Lipid Oxidation Products), page 1 of that particular unit. Many reagents and procedures are employed repeatedly throughout the manual. Instead of duplicating this information, cross-references among units are used extensively. Cross-referencing helps to ensure that lengthy and complex protocols are not overburdened with steps describing auxiliary procedures needed to prepare raw materials and analyze results. Certain units that describe commonly used techniques and recipes are cross-referenced in other units that describe their application. Introductory and Explanatory Information Because this publication is first and foremost a compilation of laboratory techniques in food analytical chemistry, we have not offered extensive instructive material. We have, however, included explanatory information where required to help readers gain an intuitive grasp of the procedures. Some chapters begin with overview units that describe the state of the art of the topic matter and provide a context for the procedures that follow. Section and unit introductions describe how the protocols that follow connect to one another, and annotations to the actual protocol steps describe what is happening as a procedure is carried out. Finally, the Commentary that closes each protocol unit describes background information regarding the historical and theoretical development of the method, as well as alternative approaches, critical parameters, troubleshooting guidelines, anticipated results, and time considerations. All units contain cited references and many indicate key references to inform users of particularly useful background reading, original descriptions, or applications of a technique. Protocols

Preface

Many units in the manual contain groups of protocols, each presented with a series of steps. The Basic Protocol is presented first in each unit and is generally the recommended or most universally applicable approach. Alternate Protocols are given where different equipment or reagents can be employed to achieve similar ends, where the starting material requires a variation in approach, or where requirements for the end product differ from those in the Basic Protocol. Support Protocols describe additional steps that are

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required to perform the Basic or Alternate Protocols; these steps are separated from the core protocol because they might be applicable to other uses in the manual, or because they are performed in a time frame separate from the Basic Protocol steps. Reagents and Solutions Reagents required for a protocol are itemized in the materials list before the procedure begins. Many are common stock solutions, others are commonly used buffers or media, whereas others are solutions unique to a particular protocol. Recipes for the latter solutions are supplied in each unit, following the protocols (and before the commentary) under the heading Reagents and Solutions. It is important to note that the names of some of these special solutions might be similar from unit to unit (e.g., SDS sample buffer) while the recipes differ; thus, make certain that reagents are prepared from the proper recipes. On the other hand, recipes for commonly used stock solutions and buffers are listed once in APPENDIX 2A. These universal recipes are cross-referenced parenthetically in the materials lists rather than repeated with every usage. Commercial Suppliers In some instances throughout the manual, we have recommended commercial suppliers of chemicals, biological materials, or equipment. This has been avoided wherever possible, because preference for a specific brand is subjective and is generally not based on extensive comparison testing. Our guidelines for recommending a supplier are that (1) the particular brand has actually been found to be of superior quality, or (2) the item is difficult to find in the marketplace. The purity of chemical reagents frequently varies with supplier. Generally reagent grade chemicals are preferred. Special care must be paid to procedures that require dry solvents. Different suppliers provide special anhydrous grade solvents which may vary in water content depending on the supplier. Addresses, phone numbers, facsimile numbers, and web addresses of all suppliers mentioned in this manual are provided in the SUPPLIERS APPENDIX. Safety Considerations Anyone carrying out these protocols will encounter hazardous or potentially hazardous materials including toxic chemicals and carcinogenic or teratogenic reagents. Most governments regulate the use of these materials; it is essential that they be used in strict accordance with local and national regulations. Cautionary notes are included in many instances throughout the manual, but we emphasize that users must proceed with the prudence and precaution associated with good laboratory practice, and that all materials be used in strict accordance with local and national regulations. Reader Response Most of the protocols included in this manual are used routinely in our own laboratories. These protocols work for us; to make them work for you we have annotated critical steps and included critical parameters and troubleshooting guides in the commentaries to most units. However, the successful evolution of this manual depends upon readers’ observations and suggestions. Consequently, a self-mailing reader-response survey can be found at the back of the manual (and is included with each supplement); we encourage readers to send in their comments.

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ACKNOWLEDGMENTS This manual is the product of dedicated efforts by many of our scientific colleagues who are acknowledged in each unit and by the hard work of the Current Protocols editorial staff at John Wiley and Sons. The publisher’s commitment and continuing support for a food analytical chemistry manual were essential for realizing this ambitious project. We are extremely grateful for the critical contributions by Ann Boyle and Elizabeth Harkins (Series Editors) who kept the editors and the contributors on track and played a key role in bringing the entire project to completion. Other skilled members of the Current Protocols staff who contributed to the project include Tom Cannon Jr., Davide Dickson, Michael Gates, Tuan Hoang, Alice Ro, Liana Scalettar, Mary Keith Trawick, and Joseph White. The extensive copyediting required to produce an accurate protocols manual was ably handled by Allen Ranz, Amy Fluet, Tom Downey, and Susan Lieberman. KEY REFERENCES Association of Official Analytical Chemists (AOAC): Official Methods of Analysis, 2000. AOAC, Arlington, Va. A compilation of analytical methods which have been collaboratively tested and approved as official methods by the AOAC. Available as a monograph, in loose-leaf binder or CD-ROM. Food Chemicals Codex. Fourth Edition, 1996. Committee on Food Chemicals Codex, National Academy of Sciences, National Research Council, Washington D.C. National Academy Press. Available on CD-ROM, CRC Press, Boca Raton, Fla. Provides quality standards for an extensive list of food chemicals along with chemical and physical methods for their determination. Journal of the Association of Official Analytical Chemists. AOAC. Arlington, Va. Peer-reviewed journal providing original articles on methods of analysis, compositional data and collaborative studies. AACC Approved Methods, 10th Edition, 2000. American Association of Cereal Chemists, St. Paul, Minn. Standardized, approved methods for analysis of cereal grains and cereal-based ingredients. Available in printed form or CD-ROM. American Journal of Enology and Viticulture. American Society of Enology and Viticulture, Davis, Calif. Journal contains peer-reviewed articles giving analytical methods for analysis of wines and grape products. American Oil Chemists Association (AOCS): Official and Tentative Methods of Analysis. American Oil Chemists Society, Champaign, Ill. A compilation of standard methods for the analysis of fats and oils approved by the American Oil Chemists Association. American Society of Brewing Chemists. Methods of Analysis of the American Society of Brewing Chemists 1992. 8th edition, ASBC, St. Paul, Minn. Standard methods of analysis recommended by the American Society of Brewing Chemists.

Ronald E. Wrolstad, Terry E. Acree, Haejung An, Eric A. Decker, Michael H. Penner, David S. Reid, Steven J. Schwartz, Charles F. Shoemaker, Denise M. Smith, and Peter Sporns

Preface

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Gravimetric Determination of Water by Drying and Weighing

UNIT A1.1

Water (moisture) in a sample is measured gravimetrically by determining the weight loss in a sample after it has been placed in an appropriate oven (convection, vacuum, or microwave) for a given time. In addition, there are automatic moisture analyzers available that utilize infrared lamps as a heat source. These types of moisture analyzers are fast but many times are matrix dependent, which requires some trial-and-error testing to determine the correct settings (power and time). Water and moisture are used interchangeably in the description of these protocols. In addition, it is assumed in the gravimetric method that only water is removed in the drying process, when in fact there may be volatile loss in some samples. Although the measurement of weight loss due to evaporation of water is frequently used to calculate moisture content, it should be pointed out that the value obtained may not be a true measure of water content. In some samples, only a proportion of the water present is lost at the drying temperature. The balance (bound water) is difficult to remove completely. In addition, the water lost may actually increase as the temperature is raised. Some samples with high fat content may exhibit volatile oil loss at drying temperatures of 100°C. Weight loss may also be dependent on such factors as particle size, weight of samples used, type of dish used, and temperature variations in the oven from shelf to shelf. Thus, it is important to compare results obtained using the same drying conditions. This unit provides three protocols for which there are established procedures for various matrices. The Basic Protocol describes water removal and quantitation after a sample is placed in a convection oven. It is probably the method of choice when one does not know which method to choose when dealing with an unknown matrix, or when one looks at samples that foam excessively in the vacuum oven method or “react,” such as popcorn under vacuum. Alternate Protocol 1 describes water removal and quantitation after a sample is placed in a vacuum oven. Because it is at reduced pressure, drying times are slightly reduced compared to the convection method. In addition, drying temperatures 5 ng/band) appear as black bands on a gray background. Sensitivity may be enhanced by brief alkali treatment of the membrane with 1% KOH followed by several rinses with PBS. Materials Blot transfer membrane (UNIT B3.2) Tween 20 solution: 0.3% (v/v) Tween 20 in PBS (prepare solution fresh weekly and store at 4°C) India ink solution: 0.1% (v/v) India ink (Pelikan 17 black) in Tween 20 solution (store 1 month at room temperature) Plastic box

Detection of Proteins on Blot Membranes

1. Place blot transfer membrane in a plastic box. Wash with water three times for 5 min each.

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Current Protocols in Food Analytical Chemistry

2. Wash membrane with Tween 20 solution four times for 10 min each. 3. Stain membrane with India ink solution for 2 hr or overnight. 4. Rinse with water until an acceptable background is obtained, then air dry. FLUORESCAMINE LABELING Fluorescamine, or 4-phenylspiro[furan-2(3H),1′-phthalan]-3,3′-dione, is used to introduce a fluorescent label on electroblotted proteins via reaction with free amines. Transferred proteins are visualized on blot transfer membranes with UV light. This stain can be very sensitive and can be used in conjunction with a second detection method such as immunoblotting (also see Basic Protocol 3). However, the protein is irreversibly modified because fluorescamine reacts with available amino groups (i.e., lysines and the protein N terminus if it was not previously blocked).

BASIC PROTOCOL 7

Materials Blot transfer membrane (UNIT B3.2) Sodium bicarbonate solution: 100 mM sodium bicarbonate in 0.3% (v/v) Tween 20, pH 9.0 (prepare fresh weekly and store at 4°C) Fluorescamine stain: 0.25 mg/ml fluorescamine (Sigma) in sodium bicarbonate solution (prepare fresh daily) Plastic box 1. Place blot transfer membrane in a plastic box. Wash with water three times for 5 min each. 2. Wash membrane with sodium bicarbonate solution twice for 10 min each. 3. Label protein bands with fluorescamine stain for 15 min. Use enough staining solution to cover the membrane completely. 4. Wash membrane with bicarbonate solution three times for 5 min each. 5. Rinse membrane several times with water. 6. Visualize transferred proteins with UV light. IAEDANS LABELING N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS or 1,5-I-AEDANS) is used for fluorescent labeling of electroblotted proteins on blot transfer membranes. Because IAEDANS reacts with free cysteines, disulfides in the sample must first be reduced with dithiothreitol (DTT). Transferred protein bands are visualized under UV light.

ALTERNATE PROTOCOL

Additional Materials (also see Basic Protocol 7) DTT solution: 200 mM dithiothreitol (DTT) in 100 mM Tris⋅Cl, pH 8.6 (APPENDIX 2A; prepare immediately before use) 100 mM Tris⋅Cl, pH 8.6 (APPENDIX 2A) N-iodoacetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (IAEDANS; Sigma; store desiccated in the dark at −20°C) Glass box 1. Place blot transfer membrane in a glass box. Wash with water three times for 5 min each. 2. Incubate membrane with DTT solution for 30 min to reduce disulfides in the sample. 3. Wash membrane with 100 mM Tris⋅Cl (pH 8.6) three times for 5 min each.

Characterization of Proteins

B3.3.5 Current Protocols in Food Analytical Chemistry

Supplement 2

This washing step probably could be deleted if proteins have been electroblotted from a reducing gel (i.e., containing DTT and/or 2-mercaptoethanol). However, it is advisable to include this simple step routinely as some oxidation may occur during electrotransfer or on the blotted membrane itself during drying or storage.

4. Dissolve 86 mg IAEDANS (2 mM final concentration) in 100 ml of 100 mM Tris⋅Cl (pH 8.6). This step should be carried out in the dark (solution should be made fresh daily).

5. Add IAEDANS solution to the membrane and allow the reaction to proceed in the dark for 30 min with shaking. 6. Wash membrane with 100 mM Tris⋅Cl (pH 8.6) twice for 5 min each. Thoroughly rinse membrane with water to remove excess reagent. 7. Visualize the transferred proteins under UV light. COMMENTARY Background Information

Detection of Proteins on Blot Membranes

The recent rapid expansion in the use of electrophoretic transfer of separated proteins to different types of membranes has necessitated the adaptation of existing protein staining techniques to transfer membranes. On-blot staining techniques serve multiple purposes, including detection of proteins for structural analysis and use in parallel with antibody reactivity to correlate precisely immunoreactivity with protein staining patterns. For the latter purpose an advantage of on-blot staining is that duplicate sections of membrane can be cut out and one section used for immunoblotting (UNIT B3.4) while a second section with duplicate lanes is stained with a general protein stain. The two pieces can then be precisely realigned. In contrast, a direct comparison of an immunoblot with a stained polyacrylamide gel is much less precise because of shrinking and swelling of the gel. The most common membranes used for electroblotting are polyvinylidene difluoride (PVDF) and nitrocellulose. PVDF membranes have become increasingly popular because they are easy to handle and store, whereas nitrocellulose membranes are brittle and tend to break easily when dry. A number of PVDF membranes are now commercially available that have subtle but important differences in protein binding properties resulting from different proprietary manufacturing processes. For example, BioRad Trans-Blot or Millipore Immobilon-PSQ PVDF membranes generally show higher protein binding capacities and higher binding affinities compared to Immobilon-P (Mozdzanowski and Speicher, 1992). Use of high-retention PVDF membranes usually results in higher and more consistent electroblotting recoveries

of most proteins than the use of either low-retention PVDF membranes or nitrocellulose. However, high-retention PVDF membranes tend to exhibit higher staining backgrounds, and it is more difficult to extract proteins or peptides from such membranes. The protocols for staining with amido black, Coomassie blue, Ponceau S, and AuroDye follow the suppliers’ recommendations. It should be noted that when staining PVDF membranes with Coomassie blue before N-terminal sequencing, omitting acetic acid from both the stain and destain solution is recommended to minimize potential extraction of protein from the membrane (Speicher, 1989). The protocol for India ink staining of electroblotted proteins is essentially that of Hancock and Tsang (1983). Different brands of India ink may be used, but staining sensitivity may vary as a result. The colloidal gold stain is the most sensitive membrane stain described here. As an alternative to commercially available colloidal gold stains, Moeremans et al. (1985) describe methods for preparing gold and iron solutions. Fluorescent labels are advantageous because they can be used not only for sequential detection methods on the same blot with minimal potential interference, but also for detection prior to protein extraction from the membrane. For example, after visualization of proteins with a fluorescent label, the blot can be photographed and specific bands marked with a pencil, either directly on the membrane or through a plastic bag. The latter method leaves a permanent indentation on the membrane. The blot can then be probed with antisera (i.e., immunoblotted; UNIT B3.4).

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Current Protocols in Food Analytical Chemistry

The protocol for fluorescamine labeling is based on the procedure described by Vera and Rivas (1988). Fluorescamine itself is not strongly fluorescent; however, when combined with the primary amines of proteins (i.e., N termini and lysine residues) it yields a highly fluorescent product. IAEDANS is an iodoacetic acid analog containing a naphthalene ring and is fluorescent under UV light. When the sample protein is reduced either prior to gel electrophoresis or with DTT after blotting, all cysteines are potentially available for reaction with IAEDANS. When the protein is not reduced, some cysteines may be involved in disulfide bonds and therefore not available for reaction. Additionally, some cysteines may be sterically inaccessible because of adsorption to the membrane and therefore will not react. An additional visualization technique for PVDF membranes is transillumination, described by Reig and Klein (1988). In that technique, the membrane is dried at room temperature, then wet with 20% methanol and viewed on a white light box. Protein bands appear as clear areas. Sensitivity is usually comparable to that of Coomassie blue staining.

Critical Parameters and Troubleshooting High-quality water (from a Milli-Q purification system or equivalent) should be used throughout these protocols. All plastic and glass boxes must be thoroughly cleaned by rinsing with water before use to avoid staining artifacts. Blot membranes should be handled by the edges only with gloves or, preferably, with forceps. This precaution is most critical for the more sensitive stains (i.e., colloidal gold, colloidal silver, and India ink). When using PVDF membranes, it is especially critical that the membrane does not dry between steps. If drying occurs, wet the PVDF membrane for 5 sec with 100% methanol, then rinse several times with water. A brief alkali treatment can enhance staining with India ink or colloidal gold. In a procedure described by Sutherland and Skerritt (1986), the membrane is washed with 1% (w/v) KOH for 5 min followed by several rinses with PBS. The alkali treatment can easily be incorporated at the beginning of the procedures if desired.

Anticipated Results Approximate detection limits and membrane compatibilities are listed in Table B3.3.1. It should be noted that detection limits may vary with gel size and the percentage of polyacry-

lamide in the gel used in preparation of the blot transfer membrane. The sensitivity of fluorescent stains is related to the number of reactive amino groups (fluorescamine) or cysteine residues (IAEDANS) present in the protein of interest.

Time Considerations The total time required for staining with amido black, Coomassie blue, and Ponceau S is 30 min to 1 hr; colloidal gold requires 4 to 6 hr; colloidal silver requires 1 hr; whereas staining with India ink requires 2 hr to overnight. Fluorescent labeling requires ∼1 hr. The optional alkali enhancement (see Critical Parameters and Troubleshooting) requires an additional 30 min at the beginning of the India ink and colloidal gold staining procedures.

Literature Cited Hancock, K. and Tsang, V.C.M. 1983. India ink staining of proteins on nitrocellulose paper. Anal. Biochem. 133:157-162. Moeremans, M., Daneels, G., and De Mey, J. 1985. Sensitive colloidal metal (gold or silver) staining of protein blots on nitrocellulose membranes. Anal. Biochem. 145:315-321. Mozdzanowski, J. and Speicher, D.W. 1992. Microsequence analysis of electroblotted proteins. Anal. Biochem. 207:11-18. Reig, J. and Klein, D. 1988. Submicrogram quantities of unstained proteins are visualized on polyvinylidene difluoride membranes by transillumination. Appl. Theor. Electrophor. 1:59-60. Speicher, D.W. 1989. Microsequencing with PVDF membranes: Efficient electroblotting, direct protein adsorption and sequencer program modifications. In Techniques in Protein Chemistry (T.E. Hugli, ed.) pp. 24-35. Academic Press, San Diego. Sutherland, M.W. and Skerritt, J.H. 1986. Alkali enhancement of protein staining on nitrocellulose. Electrophoresis 7:401-406. Vera, J.C. and Rivas, C. 1988. Fluorescent labeling of nitrocellulose-bound proteins at the nanogram level without changes in immunoreactivity. Anal. Biochem. 173:399-404.

Key References Moeremans et al., 1985. See above. Describes a method for preparation of colloidal metal stains. Vera and Rivas, 1988. See above. Describes the use of multiple detection methods.

Contributed by Sandra Harper and David W. Speicher The Wistar Institute Philadelphia, Pennsylvania

Characterization of Proteins

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Supplement 2

Immunoblot Detection

UNIT B3.4

Immunoblotting (often referred to as western blotting) is used to identify specific antigens recognized by polyclonal or monoclonal antibodies. Protein samples are solubilized, usually with sodium dodecyl sulfate (SDS) and in selected cases with reducing agents such as dithiothreitol (DTT) or 2-mercaptoethanol (2-ME); some antibody epitopes are destroyed if reducing conditions are used. Following solubilization, the material is separated by SDS-PAGE (using either one- or two-dimensional gels; UNIT B3.1). The antigens are then electrophoretically transferred in a tank or a semidry electroblotting unit to a nitrocellulose, polyvinylidene difluoride (PVDF), or nylon membrane (UNIT B3.2). When nitrocellulose or PVDF membranes are used, the process can be monitored by a reversible staining procedure with Ponceau S (UNIT B3.3). After staining, protein bands on the membrane can be photographed and/or the positions of the detected proteins can be marked with indelible ink (e.g., Paper-Mate pen). The membrane is then completely destained by soaking in water for an additional 10 min. At this point the transferred proteins are bound to the surface of the membrane, providing access for reaction with immunodetection reagents. All remaining binding sites are blocked by immersing the membrane in a solution containing either a protein or detergent blocking agent. After being probed with primary antibody, the membrane is washed and the antibody-antigen complexes are identified using horseradish peroxidase (HRP) or alkaline phosphatase (AP) enzymes coupled to the secondary anti-immunoglobulin-G (anti-IgG) antibody (e.g., goat anti-rabbit IgG). The enzymes are attached directly (Basic Protocol) or via an avidin-biotin bridge (Alternate Protocol) to the secondary antibody. Chromogenic or luminescent substrates (Support Protocols 1 and 2) are then used to visualize the activity. IMMUNOPROBING WITH DIRECTLY CONJUGATED SECONDARY ANTIBODY

BASIC PROTOCOL

After electrophoretic transfer to the membrane (UNIT B3.2), the immobilized proteins are probed with specific antibodies to identify and quantitate any antigens present. The membrane is first immersed in blocking buffer to fill all protein-binding sites with a nonreactive protein or detergent. Next, the membrane is placed in a solution containing an antibody directed against the antigen (primary antibody). The blot is washed and then exposed to an enzyme-antibody conjugate directed against the primary antibody (secondary antibody; e.g., goat anti-rabbit IgG). Antigens are identified by chromogenic or luminescent visualization (see Support Protocols 1 and 2) of the antigen/primary antibody/secondary antibody/enzyme complex bound to the membrane. Tween 20 is a common alternative to protein blocking agents for use with nitrocellulose or PVDF filters. Materials Membrane with transferred proteins (UNIT B3.2) Blocking buffer (see recipe) appropriate for membrane and detection protocol Primary antibody specific for protein of interest TTBS (nitrocellulose or PVDF) or TBS (neutral or positively charged nylon; see recipes for both solutions) Secondary antibody conjugate: horseradish peroxidase (HRP)- or alkaline phosphatase (AP)-anti-Ig (Cappel, Vector, Kirkegaard & Perry, or Sigma; dilute as indicated by manufacturer and store frozen in 25-µl aliquots until use) Characterization of Proteins Contributed by Sean Gallagher Current Protocols in Food Analytical Chemistry (2001) B3.4.1-B3.4.11 Copyright © 2001 by John Wiley & Sons, Inc.

B3.4.1 Supplement 2

Heat-sealable plastic bags Powder-free gloves Plastic box Additional reagents and equipment for chromogenic or luminescent visualization (see Support Protocol 1 or see Support Protocol 2) 1. Place membrane in heat-sealable plastic bag with 5 ml blocking buffer and seal bag. Incubate 30 min to 1 hr at room temperature with agitation on an orbital shaker or rocking platform. Usually 5 ml buffer is sufficient for two to three membranes (14 × 14–cm size). Plastic incubation trays are often used in place of heat-sealable bags, and can be especially useful when processing large numbers of strips in different primary antibody solutions.

2. Dilute primary antibody in blocking buffer. Primary antibody dilution is determined empirically but is typically 1/100 to 1/1000 for a polyclonal antibody (Fig. B3.4.1; Cooper and Paterson, 1995; Andrew and Titus, 1991a,b,c, 1993), 1/10 to 1/100 for hybridoma supernatants (Yokoyama, 1991a), and ≥1/1000 for murine ascites fluid containing monoclonal antibodies (Yokoyama, 1991b). Ten- to one-hundred-fold higher dilutions can be used with alkaline phosphatase– or luminescence–based detection systems. Both primary and secondary antibody solutions can be used at least twice, but long-term storage (i.e., >2 days at 4°C) is not recommended.

3. Open bag and pour out blocking buffer. Replace with diluted primary antibody and incubate 30 min to 1 hr at room temperature with constant agitation. Usually 5 ml diluted primary antibody solution is sufficient for two to three membranes (14 × 14–cm size). Incubation time may vary, depending on conjugate used.

1/6400

1/3200

1/1600

1/800

1/400

1/200

1/100

1/50

Serum dilution

Size (kDa) 200 116 97

Figure B3.4.1 Serial dilution of primary antibody directed against the 97-kDa catalytic subunit of the plant plasma membrane ATPase. The blot was developed with HRP-coupled avidin-biotin reagents according to the Alternate Protocol and visualized with 4-chloro-1-naphthol (4CN). Note how background improves with dilution.

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B3.4.2 Supplement 2

Current Protocols in Food Analytical Chemistry

When using plastic trays, the primary and secondary antibody solution volume should be increased to 25 to 50 ml. For membrane strips, incubation trays with individual slots are recommended. Typically, 0.5 to 1 ml solution/slot is needed.

4. Remove membrane from plastic bag with gloved hand. Place in plastic box and wash 4 times by agitating with 200 ml TTBS (nitrocellulose or PVDF) or TBS (nylon), 10 to 15 min each time. 5. Dilute secondary antibody HRP- or AP-anti-Ig conjugate in blocking buffer. Commercially available enzyme-conjugated secondary antibody is usually diluted 1/200 to 1/2000 (i.e., 20 ìl/ml to 2 ìl/ml) prior to use (Harlow and Lane, 1988).

6. Place membrane in fresh heat-sealable plastic bag, add diluted HRP- or AP-anti-Ig conjugate, and incubate 30 min to 1 hr at room temperature with constant agitation. When using plastic incubation trays, see step 3 annotation for proper antibody solution volumes.

7. Remove membrane from bag and wash as in step 4. Develop according to appropriate visualization protocol (see Support Protocol 1 or see Support Protocol 2). IMMUNOPROBING WITH AVIDIN-BIOTIN COUPLING TO SECONDARY ANTIBODY

ALTERNATE PROTOCOL

The following procedure is based on the Vectastain ABC kit from Vector (SUPPLIERS APPENDIX). It uses an avidin-biotin complex to attach horseradish peroxidase (HRP) or alkaline phosphatase (AP) to the biotinylated secondary antibody. Avidin-biotin systems are capable of extremely high sensitivity because multiple reporter enzymes are bound to each secondary antibody. In addition, the detergent Tween 20 is a popular alternative to protein blocking agents when using nitrocellulose or PVDF membranes. Additional Materials (also see Basic Protocol) Vectastain ABC (HRP) or ABC-AP (AP) kit (Vector) containing the following: reagent A (avidin), reagent B (biotinylated HRP or AP), and biotinylated secondary antibody (request membrane immunodetection protocols when ordering) 1. Equilibrate membrane in appropriate blocking buffer in heat-sealed plastic bag with constant agitation using an orbital shaker or rocking platform. For nitrocellulose and PVDF, incubate 30 to 60 min at room temperature. For nylon, incubate ≥2 hr at 37°C. TTBS is well suited for avidin-biotin systems. For nylon, protein binding agents are recommended. Because nonfat dry milk contains residual biotin that will interfere with the immunoassay, its use must be restricted to the blocking step only. Plastic incubation trays are often used in place of heat-sealable bags, and can be especially useful when processing large numbers of strips in different primary antibody solutions.

2. Prepare primary antibody solution in TTBS (nitrocellulose or PVDF) or TBS (nylon). Dilutions of sera containing primary antibody generally range from 1/100 to 1/10,000. This depends in large part on the sensitivity of the detection system. With high-sensitivity avidin-biotin systems, dilutions from 1/1000 to 1/100,000 are common. Higher dilutions can be used with AP- or luminescence-based detection systems. To determine the appropriate concentration of primary antibody, a dilution series is easily performed with membrane strips. Separate antigens on a preparative gel (i.e., with a single large sample well) and immunoblot the entire gel. Cut 2- to 4-mm strips by hand or with a membrane cutter (Schleicher & Schuell or Inotech) and incubate individual strips in a set of serial dilutions of primary antibody. The correct dilution should give low background and high specificity (Fig. B3.4.1). Characterization of Proteins

B3.4.3 Current Protocols in Food Analytical Chemsitry

Supplement 2

3. Open bag, remove blocking buffer, and add enough primary antibody solution to cover membrane. Reseal bag and incubate 30 min at room temperature with gentle rocking. When using plastic trays, the primary and secondary antibody solution volume should be increased to 25 to 50 ml. For membrane strips, incubation trays with individual slots are recommended. Typically, 0.5 to 1 ml solution/slot is needed.

4. Remove membrane from bag and place in plastic box. Wash membrane 3 times over a 15-min span in TTBS (nitrocellulose or PVDF) or TBS (nylon). Add enough TTBS or TBS to fully cover the membrane (e.g., 5 to 10 ml/strip or 25 to 50 ml/whole membrane). 5. Prepare biotinylated secondary antibody solution by diluting 2 drops biotinylated antibody with 50 to 100 ml TTBS (nitrocellulose or PVDF) or TBS (nylon). This dilution gives both high sensitivity and enough volume to easily cover a large (14 × 14–cm) membrane.

6. Transfer membrane to fresh plastic bag containing secondary antibody solution. Incubate 30 min at room temperature with slow rocking, then wash as in step 4. When using plastic incubation trays, see step 3 annotation for proper antibody solution volumes.

7. While membrane is being incubated with secondary antibody, prepare avidin-biotinHRP or -AP complex. Mix 2 drops Vectastain reagent A and 2 drops reagent B into 10 ml TTBS (nitrocellulose or PVDF) or TBS (nylon). Incubate 30 min at room temperature, then further dilute to 50 ml with TTBS or TBS. Diluting the A and B reagents to 50 ml expands the amount of membrane that can be probed without greatly affecting sensitivity. Azide is a peroxidase inhibitor and should not be used as a preservative for long-term storage of the antibody solution. Casein, nonfat dry milk, serum, and some grades of bovine serum albumin (BSA) may interfere with the formation of the avidin-biotin complex and should not be used in the presence of avidin or biotin reagents (Gillespie and Hudspeth, 1991; see also instructions from Vector).

8. Transfer membrane to fresh plastic bag containing avidin-biotin-enzyme solution. Incubate 30 min at room temperature with slow rocking, then wash over a 30-min span as in step 4. Hybridization in a plastic bag requires 5 to 10 ml avidin-biotin-enzyme solution. Membrane strips require 5 to 10 ml/strip, whereas blots from standard-sized gels (i.e., 14 × 16 cm) require 50 ml for convenient handling in a tray.

9. Develop membrane according to the appropriate visualization protocol (see Support Protocol 1 or see Support Protocol 2). SUPPORT PROTOCOL 1

VISUALIZATION WITH CHROMOGENIC SUBSTRATES After incubation with primary and secondary antibody conjugates (see Basic Protocol or see Alternate Protocol), bound antigens are typically visualized with chromogenic substrates. The substrates 4CN, DAB/NiCl2, and TMB are commonly used with horseradish peroxidase (HRP)–based immunodetection procedures, whereas BCIP/NBT is recommended for alkaline phosphatase (AP)–based procedures (see Table B3.4.1). After incubation with primary and secondary antibodies, the membrane is placed in the appropriate substrate solution. Protein bands usually appear within a few minutes. Materials Membrane with transferred proteins and probed with antibody-enzyme complex (see Basic Protocol or see Alternate Protocol) TBS (see recipe) Chromogenic visualization solution (Table B3.4.1; see recipes)

Immunoblot Detection

Additional reagents and equipment for gel photography

B3.4.4 Supplement 2

Current Protocols in Food Analytical Chemistry

1. If final membrane wash (see Basic Protocol, step 7, or see Alternate Protocol, step 9) was performed in TTBS, wash membrane 15 min at room temperature in 50 ml TBS. The Tween 20 in the TTBS interferes with 4CN development (Bjerrum et al., 1988).

2. Place membrane into chromogenic visualization solution. Bands should appear in 10 to 30 min. 3. Terminate reaction by washing membrane in distilled water. Air dry and photograph for a permanent record.

Table B3.4.1

Chromogenic and Luminescent Visualization Systemsa

System

Reagentb

Reaction/detection

Commentsc

Chromogenic HRP-based

4CN

Oxidized products form purple precipitate

DAB/NiCl2d

Forms dark brown precipitate

TMBe

Forms dark purple stain

BCIP/NBT

BCIP hydrolysis produces indigo precipitate after oxidation with NBT; reduced NBT precipitates; dark blue-gray stain results

Not very sensitive (Tween 20 inhibits reaction); fades rapidly on exposure to light More sensitive than 4CN but potentially carcinogenic; resulting membrane is easily scanned More stable and less toxic than DAB/NiCl2; may be somewhat more sensitivee; can be used with all membrane types More sensitive and reliable than other AP-precipitating substrates; note that phosphate inhibits AP activity

Luminol/H2O2/ p-iodophenol

Oxidized luminol substrate gives off blue light; p-iodophenol increases light output Dephosphorylated substrate gives off light

AP-based

Luminescent HRP-based

AP-based

Substituted 1,2dioxetane phosphates (e.g., AMPPD, CSPD, Lumigen-PPD, Lumi-Phos 530f)

Very convenient, sensitive system; reaction is detected within a few seconds to 1 hr Protocol described gives reasonable sensitivity on all membrane types; consult instructions of reagent manufacturer for maximum sensitivity and minimum background (see Troubleshooting)

aAbbreviations: AMPPD or Lumigen-PPD, disodium 3-(4-methoxyspiro{1,2-dioxetane-3,2′-tricyclo[3.3.1.13,7]-decan}-4-yl)phenyl phosphate; AP, alkaline phosphatase; BCIP, 5-bromo-4-chloro-3-indolyl phosphate; 4CN, 4-chloro-1-napthol; CSPD, AMPPD with substituted chlorine moiety on adamantine ring; DAB, 3,3′-diaminobenzidine; HRP, horseradish peroxidase; NBT, nitroblue tetrazolium; TMB, 3,3′,5,5′-tetramethylbenzidine. bRecipes and suppliers for all reagents except TMB are listed in Reagents and Solutions. Kits containing TMB are available from Kirkegaard & Perry, TSI Center for Diagnostic Products, and Vector. cSee Commentary for further details. dDAB/NiCl can be used without the nickel enhancement, but sensitivity is greatly reduced. 2 eFirst treating nitrocellulose filters with 1% dextran sulfate for 10 min in 10 mM citrate-EDTA (pH 5.0) causes TMB to precipitate onto the membrane

with a sensitivity much greater than that seen for 4CN or DAB and equal to or better than that for BCIP/NBT (McKimm-Breschkin, 1990). fLumi-Phos 530 contains dioxetane, MgCl , cetyltrimethylammonium bromide (CTAB), and fluorescent enhancer in a pH 9.6 buffer. 2

Characterization of Proteins

B3.4.5 Current Protocols in Food Analytical Chemsitry

Supplement 2

SUPPORT PROTOCOL 2

VISUALIZATION WITH LUMINESCENT SUBSTRATES After incubation with primary and secondary antibody conjugates (see Basic Protocol and see Alternate Protocol), antigens can also be visualized with luminescent substrates. Detection with light offers both greater speed and enhanced sensitivity over chromogenic and radioisotopic procedures. After the final wash, the blot is immersed in a substrate solution containing luminol for horseradish peroxidase (HRP) systems, or dioxetane phosphate for alkaline phosphatase (AP) systems, sealed in thin plastic wrap and placed firmly against film. Exposures range from a few seconds to several hours, although typically strong signals appear within a few seconds or minutes. Additional Materials (also see Support Protocol 1) Luminescent substrate buffer: 50 mM Tris⋅Cl, pH 7.5 (APPENDIX 2A; HRP), or dioxetane phosphate substrate buffer (see recipe; AP) Nitro-Block solution (AP reactions only): 5% (v/v) Nitro-Block (Applied Biosystems) in dioxetane phosphate substrate buffer (see recipe), prepared just before use Luminescent visualization solution (Table B3.4.1; see recipes) Clear plastic wrap NOTE: See Troubleshooting section for suggestions concerning optimization of the protocol, particularly when employing AP-based systems. 1. Equilibrate membrane in two 15-min washes with substrate buffer each. For blots of whole gels, use 50 ml substrate buffer; for strips, use 5 to 10 ml/strip.

2. For AP reactions using nitrocellulose or PVDF membranes: Incubate 5 min in Nitro-Block solution, followed by 5 min in substrate buffer. For blots of whole gels, use 50 ml Nitro-Block solution and substrate buffer; for strips, use 5 to 10 ml/strip. Nitro-Block enhances light output from the dioxetane substrate in reactions using AMPPD, CSPD, or Lumigen-PPD concentrate. It is required for nitrocellulose and recommended for PVDF membranes. It is not needed for Lumi-Phos 530, AP reactions on nylon membranes, or HRP–based reactions on any type of membrane. Lumi-Phos 530 is not recommended for nitrocellulose membranes.

3. Transfer membrane to luminescent visualization solution. Soak 30 sec (HRP reactions) to 5 min (AP reactions). Alternatively, lay out a square of plastic wrap and pipet 1 to 2 ml visualization solution into the middle. Place membrane on the plastic so that the visualization solution spreads out evenly from edge to edge. Fold wrap back onto membrane, seal, and proceed to step 5.

4. Remove membrane, drain, and place face down on a sheet of clear plastic wrap. Fold wrap back onto membrane and seal with tape to form a liquid-tight enclosure. To ensure an optimal image, only one layer of plastic should be present between the membrane and film. Sealable bags are an effective alternative. Moisture must not come in contact with the X-ray film.

5. In a darkroom, place membrane face down onto film. Do this quickly and do not reposition; a double image will be formed if the membrane is moved while in contact with the film. A blurred image is usually caused by poor contact between membrane and film; use a film cassette that ensures a tight fit.

6. Expose film for a few seconds to several hours. Immunoblot Detection

Typically, immunoblots produce very strong signals within a few seconds or minutes; however, weak signals may require several hours to an overnight exposure. If no image is detected, expose film 30 min to 1 hr and, if needed, overnight (see Troubleshooting).

B3.4.6 Supplement 2

Current Protocols in Food Analytical Chemistry

7. If desired, wash membrane in two 15-min washes of 50 ml TBS and process for chromogenic development (see Support Protocol 1). It is possible to develop the same membrane with chromogenic substrates after luminescent visualization.

REAGENTS AND SOLUTIONS Use Milli-Q-purified water or equivalent in all recipes and protocol steps. For common stock solutions, see APPENDIX 2A; for suppliers, see SUPPLIERS APPENDIX.

Alkaline phosphate substrate buffer 100 mM Tris⋅Cl, pH 9.5 100 mM NaCl 5 mM MgCl2 Blocking buffer Colorimetric detection For nitrocellulose and PVDF: 0.1% (v/v) Tween 20 in TBS (TTBS; see recipe). For neutral and positively charged nylon: Tris-buffered saline (TBS; see recipe) containing 10% (w/v) nonfat dry milk. TTBS can be stored up to 1 week at 4°C. Prepare blocking buffer containing nonfat dry milk immediately prior to use, as the milk blocking solution is not stable.

Luminescent detection For nitrocellulose, PVDF, and neutral nylon (e.g., Pall Biodyne A): 0.2% (w/v) casein (e.g., Hammarsten grade or I-Block; Applied Biosystems) in TTBS (see recipe). For positively charged nylon: 6% (w/v) casein/1% (v/v) polyvinylpyrrolidone (PVP) in TTBS (see recipe). For each solution: With constant mixing, add casein and PVP to warm (65°C) TTBS. Stir for 5 min, then cool. Prepare each solution just before use. Chromogenic visualization solutions BCIP/NBT visualization solution: Mix 33 µl NBT stock [100 mg NBT in 2 ml 70% (v/v) dimethylformamide (DMF), stored