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GENERATION AND APPLICATION OF IMMUNOLOGICAL PROBES FOR THE STUDY OF MEMBRANE‐BOUND PROTEINS AND ENZYMES—A REVIEW

ABSTRACT Immunological techniques are widely used for the detection of enzymes in food systems and for elucidating protein structure and function. The generation of anti‐bodies recognizing membrane‐bound proteins is difficult due to their hydrophobic nature, and reduced antigenicity. This review summarizes strategies for the production and purification of polyclonal antibodies recognizing membrane proteins. Applications such as enzyme immunoprecipitation, use of site‐specific anti‐peptide. antibodies for determination of the topology of membrane‐embedded proteins, immunocytochemistry, and expression vector cloning are described.


INTRODUCTION
Immunological research methods are widely used to address problems in microbial, plant and muscle systems that would be difficult or impossible to approach by traditional techniques. Antibodies have become essential tools in all phases of protein biochemistry. In 1970 the percentage of articles published in Zhe Journal of Cell Biology in which antibodies were used was 1%, while in 1990 that percentage rose to 70% (Drenckhahn et al. 1993).
The original methods available for the generation of antibodies were created with hydrophilic, antigenic proteins in mind. Membrane-bound proteins, however, are hydrophobic and therefore require different approaches for antibody generation. The biological membrane is an invaluable structure in the cell serving numerous functions, including protection, containment, transport, recognition, and biosynthesis (Gennis 1989). Due to the hydrophobic nature of proteins embedded within the membrane, purification, and subsequently antibody production, have been difficult to achieve. This is especially true with plant-derived membrane proteins, where many important enzymes are limited in abundance (Sussman 1 994).
Immunological procedures such as immunocytochemistry and immunoprecipitation are absolutely necessary in order to identify protein complexes associated with the membrane, therefore the generation of polyclonal or monoclonal antibodies against membrane-bound proteins lies at the heart of elucidating membrane structure and function. The objective of this review is to summarize the strategies used to generate polyclonal antibodies to membrane-bound proteins and to illustrate some of the available applications once these antibodies have been produced. While most of the selected examples cited in this review are based upon living systems, the approaches described should serve as important tools for investigating mechanisms of senescence and quality maintenance in postharvest and postmortem food systems (Haard 1995). Some examples of important membrane-bound proteins which occur in edible tissue are summarized in Table  1. Diagnostic membrane-associated marker enzymes used in subcellular fractionation studies to identify plant organelles are listed in Table 2.

Classes of Membrane-Bound Proteins
Proteins and lipids comprise the major components of the biological membranes of living systems. Proteins occur in varying amounts, ranging from about 20% to 80% dry weight (Gennis 1989). Membrane-localized proteins serve as enzymes, transporters, receptors, and pores. Individual membrane proteins are distinguished by their primary structures, as well as by post-translational modifications such as glycosylation (Faye et al. 1993;Herscovics and Orlean 1993), prenylation (Maltese 1990) and glycophosphatidylinositol anchoring (Cross 1990;Takeda and Kinoshita 1995).
Membrane-bound proteins are classified as either integral or peripheral. Integral membrane proteins are largely embedded in the membrane and are therefore strongly hydrophobic. Detergents must be used to solubilize integral membrane proteins from the membrane (Hjelmeland and Chrambach 1984). Integral proteins virtually always contain multiple transmembrane segments, connected by hydrophilic sequences which loop out of the membrane bilayer (Gennis 1989). The number of transmembrane helices and surface loops is determined by hydropathy analysis, which requires a full-length cDNA clone (Kyte and Doolit- . Peripherally-associated membrane proteins can be hydrophobic or hydrophilic but are not embedded within the membrane. They associate with the membrane by either electrostatic or hydrophobic interactions, or via a hydrophobic "tail" anchored in the membrane, and can be dissociated from the membrane by washing with salts or chaotropic ions (Gennis 1989). Some membrane-bound proteins, such as plasma membrane H + -ATPases (Ser-ran0 1989;Sussman 1994), members of the major intrinsic protein (MIP) family (Chrispeels and Maurel 1994;Agre et al. 1995;King and Agre 1996;Verkman etal. 1996) and the vacuolar H+-pyrophosphatase of higher plants (Rea etal. 1992;Kim et al. 1994) exist as individual subunits or multisubunit complexes embedded largely within the membrane. Onthe other hand, large multisubunit complexes such as the chloroplast ATPase (Feng and McCarty 1990a;Feng and McCarty 1990b;Avital and Gromet-Elhanan 1991), chloroplast photosystems (Cohen et al. 1995), coated vesicle ATPase (Arai et al. 1988;Adachi et al. 1990a;Adachi et al. 1990b) and tonoplast ATPase of plants (Rea et al. 1987;Lai et al. 1988;Parry et al. 1989;Sze et al. 1992) and fungi (Bowman et al. 1989;Moriyama and Nelson 1989) contain a combination of integral and peripheral subunits, and can be visualized by "ball and stalk-like'' protrusions from the membrane (Dschida and Bowman 1992;Getz and Klein 1995). Examples of well-characterized peripherally-bound proteins would include the plasma membrane GTP-binding subunits (Rho lp) of yeast (1,3)-@-glucan synthases (Drgonova et al. 1996;Qadota et al. 1996), and a 62-kDa sucrose binding protein associated with the external surface the plasma membrane of soybean (Overvoorde and Grimes 1994;Overvoorde et al. 1996).
Since membrane-bound proteins are often large multisubunit enzyme complexes, it is often necessary to dissociate the complex and generate antibodies against individual subunits before immunological studies can be conducted. The presence of detergent to prevent aggregation and to maintain the protein in a solubilized state is necessary at all times when preparing antigen, as well as the appropriate reducing agent to maintain dissociated subunits. Oftentimes, however, antibodies produced against unfolded or partially unfolded membrane proteins do not recognize target proteins in their native-folded conformations. In these cases, uses of the antibodies is generally limited to diagnostic analysis using immunoblots.

Sources of Antigens
Native Purified Protein. Proteins used for immunization must be of high purity to avoid generation of antibodies against contaminating antigens. An adjuvant is added to the protein, which acts to stimulate the immune response nonspecifically. The antigen, coupled to a carrier protein, in the adjuvant mixture is injected into an animal, most often a rabbit. To obtain the antibody, the animal is bled at various intervals and serum is then purified (Harlow and Lane 1988;Dunbar and Schwoebel 1990). This procedure has proven successful for an increasing number of membranebound proteins found in the tonoplast, or vacuolar membrane. For example, antibody was raised against a purified preparation of a 23-kDa major integral protein of the vacuolar membrane (VM 23) of radish (Maeshima 1992). This same group previously produced an antibody raised in rabbit against total peak chromatographic fractions of a 73-kDa H + -translocating inorganic pyrophosphatase found in the vacuolar membrane of mung bean hypocotyl tissue (Maeshima and Yoshida 1989). Working with maize tonoplast H+ -ATPase, Mandala and Taiz (1986) prepared antisera using partially purified ATPase from sucrose gradient centrifugation peak fractions.
In the case of membrane-bound proteins the transmembrane domain contains hydrophobic residues which reduce antigenicity, deeming successful production of antibody against an intact hydrophobic protein unlikely. Separate studies conducted by Dr. Emil R. Unanue and Dr. Howard M. Grey have led to the discovery of antigen processing, whereby extracellular proteins must first be ingested and fragmented into peptides by an antigen-presenting cell before an immune response can occur (Engelhard 1994). Consequently, it should be possible to carry out peptide digestion with an insoluble enzyme such as trypsin attached to polyacrylamide beads in order to improve antigenicity. Short peptides containing the hydrophilic loop portions of the protein may be produced during trypsinization. Since the trypsin is insoluble, it can be centrifuged, pelleted, and removed, leaving only the fragmented protein. Trypsinized peptides can then be used as an antigen for immunization. This approach was used to generate antibodies recognizing plasma membrane intrinsic proteins from Beta vulgaris L. (Wasserman er al. 1996).

SDSPAGE Gel Slices.
If an antigen is not available in pure form, it can be purified further by separation on denaturing polyacrylamide gels, excised and electroeluted. Antigens purified in this way often induce good immunological responses. The resulting antibody is useful for techniques that need or benefit from denaturation-specific antibodies (Harlow and Lane 1988).
Several different approaches exist for injection of protein from gel slices. After an SDS gel is run, the band representing the protein of interest is cut out. The gel is then fragmented by passing it repeatedly through a syringe, emulsified with adjuvant, and injected into the animal as with the native protein (Harlow and Lane 1988). This procedure works only with larger animals, such as rabbits. In order to prepare antibodies to maize tonoplast ATPase, Coomassie-stained gel slices of both 62 kDa and 72 kDa proteins were excised, crushed with buffer in a glass homogenizer, and then mixed with Freund's adjuvant and injected into New Zealand White rabbits (Mandala and Taiz 1986).
In another procedure gel slices are dried, ground into a powder, and then resuspended for injection (Harlow and Lane 1988), as was done with a 62-kDa band corresponding to a membrane protein associated with sucrose transport in soybean (Ripp et al. 1988).
Proteins can also be electroeluted from the gel in the presence of reducing agent as a further purification step and used to immunize the host animal (Harlow and Lane 1988). This method has been utilized for the generation of antibodies to several membrane-bound proteins, such as TP 25, an abundant vacuolar integral membrane protein from soybean (Johnson et al. 1989), peroxisome membrane proteins in oilseed glyoxysomes (Corpas et al. 1994), and a 43 kDa polypeptide located in heart gap junctions (Yancey el al. 1989).
SiteSpecific Anti-Peptide Antibodies. As more DNA sequences and their corresponding protein sequences have become available due to technological advances such as peptide sequencing and PCR, synthetic or microbially-expressed peptides are now increasingly used. These antibodies can be targeted against specific domains of a protein. Thus, many previously uncharacterized proteins are now amenable to topological analysis. These peptides are synthesized by Merrifield solid-phase techniques, or produced genetically. Once obtained, the purified antigens can be coupled to carrier proteins, and injected into an animal. An antibody produced in this manner is termed an anti-peptide antibody (Harlow and Lane 1988).
To carry out this procedure, a synthetic peptide of at least ten residues must be constructed. The longer the peptide, the greater the chance of obtaining an antibody which reacts with the native protein. A peptide synthesized towards the Nor C-terminal end of a protein sequence is particularly immunogenic and will usually produce antibodies recognizing the native protein (Drenckhah et al. 1993). Several systems utilizing peptides with polyhistidine tails to facilitate purification by Ni2 i . -affinity chromatography prior to immunization are available commercially.
Two important advantages of site-specific anti-peptide antibodies are that they can be prepared immediately after determining the amino acid sequence of a protein, and that particular regions of a protein can be targeted specifically for antibody production. A number of disadvantages pertaining to use of site-specific anti-peptide antibodies should be noted. First, they may not recognize the native protein if certain domains are deeply embedded in the membrane and inaccessi-ble to the antibody. This means that they may be unable to immunoprecipitate enzyme activity and can therefore be used only for immunoblotting. In addition, due to their small size, the peptides may not possess sufficient immunogenicity to induce a satisfactory immune response. In this case, they must be coupled to carrier proteins (Harlow and Lane 1988).
An example of a synthetic peptide coupled to a carrier protein before immunization is P40, a synthetic peptide corresponding to amino acid residues 40-50 of the beef heart ADPIATP carrier from mitochondria (Marty et al. 1992). This peptide was synthesized by the Merrifield solid-state technique and coupled to an ovalbumin carrier protein prior to injection. Similarly, a peptide corresponding to nine amino acid residues of a K+-conducting protein in chloroplasts (Mi et ul. 1994) was synthetically conjugated to maleimide-activated keyhole limpet hemacyanin (Tsao et ul. 1991).
Bacterial Overexpression Vectors. When antibodies are needed against a protein for which a cloned gene is available, bacterial overexpression systems represent an excellent antigen source. Coding regions may be expressed in bacteria either alone or as fusion proteins. Vectors producing glutathione S-transferase, 0-galactosidase or tryptophan fusion proteins are the most commonly used. Several vectors are commercially available for this purpose.
An antibody was raised against tonoplast intrinsic protein (TIP) from Arabidopsis thulium using a glutathione S-transferase-y-TIP fusion construct introduced into Escherichia coli (Hofte et al. 1992). This culture was then grown, and the fusion protein isolated and purified by centrifugation and gel electrophoresis. Electroeluted protein from SDS polyacrylamide gels served as antigen, and antiserum was prepared in rabbit. Antibodies against water channel proteins in the A. thulium plasma membrane (PIP1 and PIE) were generated from a fragment of PIPla encoding the 42 N-terminal amino acids cloned into a plasmid (Kammerloher et af. 1994). This fusion protein was then used to affinity-purify specific anti-PIP1 antibodies from the anti-plasma membrane serum, and to characterize the subcellular location of the PIP proteins. Amnity Purification. Crude immune serum often contains contaminating antibodies which may cross-react with proteins other than the targeted antigen. Ammonium sulfate precipitation is one of the most common methods for partial purification of antibodies (Harlow and Lane 1988;Dunbar and Schwoebel 1990). Saturated ammonium sulfate is added to a batch volume of antiserum while stirring. The mixture is then centrifuged, and the pellet resuspended and dialyzed to remove the salt. This method does not yield an antibody of high purity, but is convenient for large volumes.
Affinity columns may be used to yield pure antibodies. The crude antiserum is passed through a column to which is bound protein A-, antigen-, or antiimmunoglobulin-coupled beads. Elution occurs at low pH. The diluted purified serum is then concentrated and stored in sodium azide (Harlow and Lane 1988). Blot-affinity purification is a simple method to rid serum of contaminating antibodies (Olmsted 1981; Smith and Fisher 1984;Mishkind et al. 1987;Tang 1993). This method utilizes antigen-containing nitrocellulose strips to capture and purify antibodies recognizing a specific polypeptide. In the first step purified antigen is run on SDS-PAGE. Following electrotransfer to nitrocellulose, horizontal strips corresponding to desired antigens are excised from the membrane. Antibodies are then captured, eluted and neutralized. A desireable feature of the blot-affinity purification procedure is that both crude serum and the nitrocellulose strips are reusable.

Applications
Once specific antibodies have been produced against a membrane-bound protein, they can be used: (1) To associate an integral membrane protein with a corresponding enzymatic activity by immunoprecipitation.
(2) To probe membrane topology using vesicles of defined sidedness. (3) To determine the subcellular localization of a protein using immunocytochemistry. (4) To probe cDNA expression libraries to immunoselect clones encoding these proteins. Use of preimmune serum as a control to ensure against nonspecific binding and thus artifactual results is imperative to any of these applications.
Immunoprecipitation. Immunoprecipitation has various uses, including the determination of the molecular mass of an antigen, characterization of the specificity of an antibody, and identification of molecules associated with antigens (Otto and Lee 1993). In principle, an enzyme preparation is incubated with antiserum generated against the polypeptide of interest. If the antigen-antibody complex cannot be pulled down by centrifugation alone, a secondary agent which has affinity for IgG, such as protein A coupled to agarose beads, is added to effect precipitation. Supernatants are then assayed to detect any loss in enzyme activity, or the immunoprecipitates are then analyzed for a dose-dependent increase in the corresponding protein band.
The use of polyclonal antibodies is advantageous in that they bind to multiple sites on the antigen and provide multiple binding sites for protein A molecules, which effects precipitation of antigen-antibody complexes. These multiple interactions provide an extremely stable complex (Harlow and Lane 1988). However, in the case of membrane proteins, it must be ensured that the protein remains solubilized. If the protein were to aggregate in the absence of adequate detergent, it could be pelleted during the centrifugation step of the reaction and falsely show a loss in activity. Effective solubilizing agents for each membrane-bound protein differ, necessitating a sound characterization of the enzyme system. Furthermore, membrane proteins tend to be very "sticky" and may bind nonspecifically to the protein A-agarose. Therefore, proper control samples must be prepared to ensure the protein is precipitated due to antigen-antibody interactions.
Once the immunoprecipitation reaction has occurred, pellet fractions can be analyzed by SDS-PAGE. Potato membrane protein pp34 was immunoprecipitated using protein A-Sepharose in Nonidet P 4 0 buffer and the immunoadsorbed proteins were analyzed using SDS-PAGE to visualize the presence of the 34-kDa band (Jacinto er ul. 1993). Neurosporu crmsu plasma membrane H+-ATPase activity was neutralized using protein A-Sepharose, but in Triton X-100 buffer (Lin and Addison 1994). Fusion proteins corresponding to different transmembrane segments of the ATPase were incubated with polyclonal antibodies directed against either the Nor C-terminus. Immunoprecipitation and subsequent SDS-PAGE analysis enabled the authors to discern that the carboxyl terminus was extracytoplasmic and the amino terminus was on the cytoplasmic side of microsomal membranes.
The effects of immunoprecipitation can also be quantified by assaying an aliquot of the supernatant after centrifugation and observing a significant decrease in enzyme activity. Glucan synthase II probed with antibodies to 55 and 70 kDa polypeptides showed a decrease in activity at increasing concentrations of IgG (Dhugga and Ray 1994). Antibodies raised against partially purified (1,3)-/3-Dglucan synthase from Saccharomyces cerevisiue were able to immunoprecipitate enzyme activity and react with a 200-kDa polypeptide on Western blots, implicating the 200-kDa polypeptide as a subunit of yeast glucan synthase (Inoue et al. 1995).
Simple addition of antisera to an assay mixture without performing the precipitation reaction is another way to observe the binding of antibodies to an enzyme. An aliquot of antibody to maize tonoplast ATPase was incubated with purified ATPase, then assayed for activity. A dose-dependent neutralization of activity was observed (Mandala and Taiz 1986).
Membrane Topology. Membrane vesicles of defined sidedness produced by freeze-thaw techniques can be useful for the determination of membrane topology. Antibodies generated against a specific region of the protein are incubated with either inside-side out (IOV) or right-side out vesicles (ROV) to localize a protein domain to a defined region on the membrane. This procedure has been used to identify the orientation of both the N-and C-termini (Botfield and Wilson 1989;Kerppola and Ames 1992) of membrane proteins. In addition, limited proteolysis of membrane vesicles followed by analysis of digestion products by immunodetection (Marty et ul. 1992) can similarly identify membrane topology. Figure 1 illustrates the use of limited proteolysis to probe the topology of plasma membrane intrinsic proteins (PMIPs) in isolated vesicles from red beet storage tissue. In an earlier study, plasma membrane proteins of 31-and 27-kDa were established as members of the major intrinsic protein (MIP) superfamily (Qi el al. 1995). To establish if these proteins were proteolyzed similarly or differentially, affinity purified antibodies to each species were produced. This study enabled the identification of specific fragmentation products derived from PMIPs 31 and 27 following hydrolysis by Pronase E (Fig. 1). To examine the effect of partially permeabilizing the vesicles, protease digestions were conducted in the absence and presence of digitonin.
Several key observations are noted. First, PMIP3 1 was resistant to proteolysis in the absence of detergent (Fig. lA, lanes 2 , 4 and 6). In contrast, PMIP27 formed a digitonin-independent proteolytic fragment of 25 kDa (Fig. lB, lanes 2, 4 and   6), and additional fragments at 20 and 21 kDa. Secondly, addition of digitonin caused a dose-dependent decrease in PMIP3 1, with its eventual complete disap- pearance at 40 pg mL-of Pronase E (Fig. lA, lanes 3, 5 and 7). These results demonstrated that PMIPs 3 1 and 27 each yield unique proteolytic fragmentation patterns and are either structurally distinct, or that they each reside in specialized domains of the plasma membrane.
The topology of fungal plasma membrane H + -ATPase was investigated using limited proteolysis of sided vesicles. The N-and C-termini of the N. crassu plasma membrane H + -ATPase were determined to be cytoplasmically located by trypsinking reconstituted cytoplasmic-side out proteoliposomes (Hennessey and Scarborough 1990). In S. cerevisiue, the binding of C-terminal antibodies to H + -ATPase in 60% ROVs increased in the presence of Tween-20 at low levels (Monk et ul. 1991). These results confirmed the cytoplasmic orientation of the C-terminus.
To investigate the topology of the melibiose carrier of E. coli. membrane vesicles were incubated with an antibody (anti-MBct 10) generated against a synthetic pep tide corresponding to the C-terminus of the melibiose carrier (3otfield and Wilson 1989). Proposed models had suggested the presence of either ten or eleven transmembrane domains, causing uncertainty as to the orientation of the Cterminus. After incubation with anti-MBct 10, membrane suspensions were centrifuged and pellets were resuspended in TBS buffer containing [1251]-protein A. Following a 2 h incubation, the mixtures were again centrihged and pellets washed in buffer. Radioactivity was quantified, and it was found that only IOVs bound anti-MBct 10, suggesting a cytoplasmic orientation of the C-terminus and supporting a topological model for the melibiose carrier with an even number of transmembrane domains.
Antibodies raised against the C-termini of hydrophobic components (HisQ and HisM) of the histidine periplasmic permease were incubated with ROVs and IOVs and their binding measured radioactively, using [IZsI] (Kerppola and Ames 1992).
The antisera preferentially bound to IOVs, exhibiting binding to ROVs only 10-20% of the level of IOV binding. These results indicate that HisQ and HisM are located on the cytoplasmic side of the membrane.
The topology of the beef heart ADP/ATP carrier in the inner mitochondrial membrane was explored using proteoly sis and immunodetection of inside-out submitochondrial particles and mitoplasts, whose inner membranes are in the rightside-out conformation (Marty er al. 1992). These mitochondrial particles were digested with a range of proteases. The cleavage products were separated by SDS-PAGE, transferred to nitrocellulose and probed with antibodies raised against a peptide corresponding to the N-terminal point of the ADP/ATP carrier chain. The membrane-bound carrier in mitochondria devoid of an outer membrane (mitoplasts) reacted with the antibodies, while inside out submitochondrial particles did not. This suggests that the N-terminus of the protein is exposed to the cytosol.
The membrane topology of aquaporin-1 (AQP-1) was assessed using limited proteolysis of Xenopus luevis oocyte membranes in an inside-out conformation (Preston et al. 1994). A proposed model had suggested the presence of six transmembrane domains with five connecting loops (Preston and Agre 1991). An epitope from the coronavirus El glycoprotein was inserted into AQP-1 at various locations on the loops. Its specific orientation was determined by proteolytic digestion of intact oocytes or inside-out membrane vesicles. Membranes from oocytes expressing wild-type AQP-1 or an El epitope-tagged AQP-1 were isolated and digested with a-chymotrypsin. Results were analyzed using SDS-PAGE and immunoblotting with an anti-N-terminal peptide. By observing which epitope-tagged regions were protease sensitive, it was found that both the N-and C-termini of AQP-1 were intracellular (Smith and Agre 1991;Nielsen et ul. 1993), indicating that three connecting loops are extracellular and two are cytoplasmic.
Immunocytochemistry. Cells can be incubated with antibodies and visualized using electron or light microscopy in order to determine the presence and subcellular localization of membrane constituents. Briefly, the tissue to be stained is embedded into a solid support, cells are fixed by cross linking, and permeabilized to allow free access to labeled antibodies. After incubation with antibodies and removal of unbound antibody, labeled tissue is visualized (Harlow and Lane 1988).
Two methods of immunostaining are immunogold labeling and immunofluorescence. Gold staining allows for high resolution, is permanent and can be visualized with either a light or electron microscope. Photochemical silver amplification must be used for high sensitivity at the light microscopy level. Colloidal gold particles bind tightly to proteins at pH values close to the PI. Gold particles are conjugated to anti-immunoglobulin antibodies, protein A, or streptavidin (Harlow and Lane 1988).
Immunogold electron microscopy was used to determine the distribution of acyl carrier protein (ACP) in E. coli and plant tissues (Slabas and Smith 1988). Antibody-labeled tissue was detected using goat anti-rabbit/colloidal gold. ACP was localized to the cytoplasm of E. coli and not to membranes. However, in rape leaf 80% of ACP was localized to the chloroplast thylakoid membrane.
The subcellular localization of a tonoplast integral membrane protein of 25 kDa (TP 25) from soybean was investigated using immunogold labeling (Johnson et al. 1989). Tonoplast membranes, but not the vacuolar matrix, were heavily labeled with gold particles following incubation with TP 25 antiserum and subsequent treatment with a colloidal-gold-coupled secondary antibody.
Immunolocalization of the plasma membrane H + -ATPase in leaves of broad bean was determined using colloidal gold particles (Bouche-Pillon et al. 1994). The antibody used for incubation was generated against an isoform of the A.
thaliana HC-ATPase expressed in E. coli. The density of gold particles was greatest on the plasma membrane of transfer cells and least dense on the plasma membrane of sieve tube, phloem parenchyma cells, and bundle sheath cells, localizing the proton pump to transfer cell membranes.
Immunofluorescence has the advantage of high resolution and the possibility to stain live cells. However, a specially equipped microscope is required and the signal is lost over time (Harlow and Lane 1988). In addition, this technique is incompatible with most histochemical stains, which autofluoresce. Fluomchromes can be conjugated to the same reagents as colloidal gold particles. AQP-3, a water channel of kidney collecting ducts, has been found to transport urea and glycerol in addition to water (Ishibashi ef al. 1994). An antibody raised against a synthetic peptide corresponding to the C-terminus of AQP-3 was incubated with sections of rat kidney fixed onto slides. After treatment with a fluorochrome conjugate and visualization, staining was detected only at the basolateral membranes of collecting duct cells and not in other nephron segments.
Immunofluorescence has been widely employed to investigate the subcellular localization of membrane-bound proteins. A glycerol intrinsic protein (GLIP) was localized to the basolateral membrane of kidney collecting duct cells and brain meningeal cells by immunofluorescence techniques (Ma et al. 1994). The polyclonal GLIP antibody was generated against a synthetic C-terminal peptide. The membrane topology of the (Na+, K + )-ATPase was investigated by epitopetagging the N-and C-termini of both the a-and / 3 subunits (Yoon and Guidotti 1994). The orientation of the epitopes was determined by immunofluorescence with cells expressing the (Na' , K + )-ATPase. The localization of the C-terminus of the yeast plasma membrane H+ -ATPase to the cytoplasmic side of the membrane was determined by immunofluorescence of isolated plasma membrane vesicles (Monk et al. 1991). The antibody was prepared against the C-terminus of the yeast ATPase.
Immunoselection. Antibodies can be used as probes to screen cDNA expression libraries when conventional cloning methods are unsuccessful. Those antibodies that recognize a short linear sequence in both unfolded and folded structures are most useful in this technique (Obar and Holzbaur 1993). Either bacterial (Zimniak et al. 1988;Sarafian er al. 1992) or mammalian (Kammerloher et al. 1994) expression systems can be employed. Following primary and secondary screening, positive clones are identified, isolated and characterized.
The carrot vacuolar H + -ATPase contains a 69 kDa subunit which is closely related to the &chain of F@,-ATPases and is thought to contain the catalytic site (Zimniak et al. 1988). A carrot root Xgtl 1 cDNA library was screened with antibodies generated against the purified 72-kDa subunit of the tonoplast H + -ATPase of corn coleoptiles. Five clones were isolated and reacted positively with antibody to the native as well as to the denatured enzyme. These positive clones were plaque affinity purified and the eluted antibodies used for immunodetection of corn tonoplast proteins. The antibodies for three different positive clones specifically bound to the 70-kDa band on Western blots. The clones were then sequenced and it was confmed that the 70-kDa subunits of plant, fungal and vacuolar ATPases are highly conserved.
To clone cDNAs encoding the A. thulium tonoplast H + -translocating inorganic pyrophosphatase (PPase), an A. thaliana cDNA expression library in AZAP was screened with affinity-purified antibodies generated against tonoplast H + -PPase purified from etiolated mung bean hypocotyls . A primary partial clone was obtained and then used to screen a cDNA library constructed in AgtlO for the isolation of a near-full-length clone. Sequence information of the tonoplast H + -PPase revealed no homology with other characterized ion translocases, suggesting a novel category of H + -translocase. Bacterial expression systems often do not provide correct folding and high expression levels of hydrophobic proteins. Therefore, mammalian COS cell expression systems have been developed when working with integral plasma membrane proteins. In order to identify genes encoding plant integral plasma membrane proteins, COS cells were transfected using a DEAE-dextran method with an A. thalianu root cDNA library (Kammerloher et al. 1994). Antibodies raised against purified deglycosylated integral plasma membrane proteins from A. thulium roots were used to screen the mammalian library. Sequence analysis of positive clones revealed several genes encoding a family of five related proteins. Antibodies immunopurified against a 27-kDa plasma membrane antigen detected each of the five proteins when individually expressed in COS cells. These proteins were determined by sequence comparisons to belong to the MIP superfamily.

SUMMARY
Due to their hydrophobicity and complexity, membrane-bound proteins, require distinct methods of purification and characterization. The application of immunological techniques is the best way to unequivocally identify the subcellular localization, membrane topology and association with enzyme activities. In addition, antibodies may be used as probes to screen cDNA libraries when traditional methods are unsuccessful. The most important aspect of using an immunological approach to a problem is the generation of an antibody which is specific, free of contaminants and of high titer. Once pure antibodies have been obtained against either an integral membrane protein or specific regions of the protein, a myriad of studies including immunoaffinity purification, expressionvector cloning and immunolocalization, can be conducted.