CHAPTER 8

Isolation and Analysis of Proteins

INTRODUCTION

M any proteins that are involved in the immune response and other biological sys-tems are initially described by reactivity with antibodies. These include proteins

identified by monoclonal antibodies (CD3; Borst et al., 1983; APPENDIX 4) or antipeptideantibodies (mouse TCR-γδ; Brenner et al., 1986; Lew et al., 1986), or by their associationwith proteins previously characterized by antibody reactivity (CD3 ζ chains; Weissmanet al., 1986). The exquisite specificity of antibodies makes them powerful tools for identi-fying, isolating, and characterizing their protein ligands, which is the primary focus of thischapter. Many of the methods, however, are not exclusive to immunochemistry—namely,the procedures involving solubilization, electrophoresis, and detection.

Selection of the appropriate methods for isolating and analyzing proteins involved in theimmune system depends on the experimental questions being asked. For example, exper-imental approaches for the analysis of integral cell membrane proteins require that suchproteins be solubilized with detergents (UNIT 8.1A) prior to isolation by antibodies (UNITS 8.2

& 8.3). Often, the cells of interest need to first be fractionated from tissues (UNIT 8.1B).Finally, some experimental questions involve the isolation and analysis of proteins as-sociated with specific organelles, thus necessitating the subcellular fractionation of cellsunder study (UNIT 8.1C).

Slight variations on the solubilization procedures (UNITS 8.2 & 8.3), such as the detergentsutilized, have been worked out for particular proteins in individual laboratories. For anuncharacterized protein, the investigator must empirically determine the procedure thatis most suitable, realizing that a number of solubilization procedures will probably yieldapproximately the same results. For example, some laboratories consider the detergentsNP-40 and Triton X-100 to be interchangeable, while others do not. In some cases, sub-sequent analytical procedures may be enhanced by isolating membranes before detergentsolubilization (UNIT 8.1A). Chemical cross-linking prior to solubilization is useful for de-termining both spatial relationships of proteins within membranes and ligand-receptorinteractions (UNIT 8.1A).

A variety of methods exist for isolating proteins, including various types of columnchromatography. Such columns have classically been run under low pressure, but nowhigher pressure is more commonly employed by means of high-performance liquid chro-matography (HPLC). In this chapter, procedures for isolating relatively large amounts ofprotein by immunoaffinity chromatography are detailed (UNIT 8.2). When working on ananalytical scale, this procedure can be scaled down to the microcentrifuge tube level withone of a variety of immunoprecipitation procedures (UNIT 8.3). UNIT 8.7 describes basicapproaches to immunoprecipitating tagged proteins from whole cell extracts. Coprecip-itation of proteins from whole-cell extracts is a valuable approach to test for physicalinteractions between proteins of interest. In a typical experiment, as described in this unit,cells are lysed and a whole-cell extract is prepared under non-denaturing conditions. Theprotein is precipitated from the lysate with a solid-phase affinity matrix, and the precip-itate is tested for the presence of a second specifically associated protein. The approachcan be used for native or epitope-tagged proteins for which antibodies are available, or forrecombinant proteins that have been engineered to bind with high affinity to a moleculethat can be coupled to a solid-phase matrix.

Current Protocols in Immunology 8.0.1-8.0.3, November 2008Published online November 2008 in Wiley Interscience (www.interscience.wiley.com).DOI: 10.1002/0471142735.im0800s83Copyright C© 2008 John Wiley & Sons, Inc.

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Once the protein is isolated, its purity and molecular properties are of interest—i.e.,molecular size, subunit composition, isoelectric point, and possibly primary structure.The purity of isolated proteins is usually assessed by electrophoresis because of superiorresolving power. For a protein without subunits or a protein with identical subunits,detection of a single protein band after one-dimensional gel electrophoresis (UNIT 8.4) ora single spot after two-dimensional gel electrophoresis (UNIT 8.5) suggests that the proteinis pure. Proteins composed of disulfide-linked subunits should yield a single band underdenaturing conditions without reducing agent. If protein subunits are noncovalently asso-ciated, a single band should be obtained under nondenaturing conditions (UNIT 8.4). Thus,by comparing results from denaturing gels with and without reducing agent, and/or fromdenaturing versus nondenaturing gels, information about purity and subunit compositionis obtained. An elegant, complementary electrophoretic procedure for investigating thesubunit composition of proteins is diagonal gel electrophoresis (UNIT 8.6). This procedurehas proven particularly useful in the analysis of T cell receptors and the componentchains of the CD3 complex (Lew et al., 1986; Weissman et al., 1986).

A critical factor in analyzing proteins on polyacrylamide gels is the means of detection.The most commonly used detection procedure is Coomassie blue staining, although sen-sitivity is gained by using silver staining (UNIT 8.9). To determine if a protein correspondsto a particular antigen as defined by antibodies, immunoblotting (i.e., western blotting)is performed (UNIT 8.10). Staining of blot transfer membranes with protein stains permitsvisualization of proteins and allows the extent of transfer to be monitored. In the protocolsdescribed in UNIT 8.10B, proteins are stained after electroblotting from one-dimensional ortwo-dimensional polyacrylamide gels to blot membranes such as polyvinylidene difluo-ride (PVDF), nitrocellulose, or nylon membranes. This is accomplished using one of sixgeneral protein stains: Amido black, Coomassie blue, Ponceau S, colloidal gold, colloidalsilver, and India ink. In addition, the fluorescent stains fluorescamine and IAEDANS,which covalently react with bound proteins, are described. Approximate detection limitsfor each nonfluorescent stain are indicated along with membrane compatibilities.

When working with small amounts of material, sensitivity of detection is enhancedthrough the use of radiolabeling (UNITS 8.11 & 8.12). Iodinated proteins are also usedas standards in radioimmunoassays, and iodinated antibodies (UNIT 8.11) or protein A(iodinated as for Ig in UNIT 8.11 or purchased from Amersham) are used as probes toidentify specific antigen-antibody complexes on cell surfaces or in ELISAs (UNIT 2.1).To obtain high specific activity, soluble proteins are usually labeled with 125I usingchloramine T catalysis (UNIT 8.11). For labeling cell-surface proteins, enzymatic oxidationwith lactoperoxidase (UNIT 8.11) or chemical catalysis with Iodogen beads (Pierce) isusually employed. Chloramine T oxidation can be used for labeling cells with iodine, butintracellular proteins are also labeled.

Nonradioactive methods for labeling membrane-bound and soluble proteins with sulfo-N-hydroxusuccinimide-biotin (sulfo-NHS-biotin) are described in UNIT 8.16. This approachoffers other practical advantages since biotinylation allows isolation of proteins fromcomplex cell lysates by immobilization on streptavidin agarose. The chemical propertiesof sulfo-NHS-biotin also prevent it from crossing the plasma membrane. In addition, theNHS group allows for derivitization of the ε-amino group of exposed lysine residues,which are twice as abundant as tyrosine residues. Thus, a wider variety of protein anda greater number of residues per molecule will be labeled with biotin as compared withthe lactoperoxidase-catalyzed method that iodinates tyrosine residues.

Biosynthetic labeling of proteins with radioactive amino acids, usually [35S]methionineand/or [35S]cysteine, is used to study the synthesis, processing, intracellular transport,secretion, and degradation of proteins (UNIT 8.12). It is also employed for radiolabeling

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cell-surface proteins in cases where proteins may not contain tyrosine residues that areaccessible to radioiodination.

Proteins can be characterized further by analyzing the content and nature of their non–amino acid components, such as phosphates (UNITS 11.2 & 11.3), sulfates, fatty acids,and, in particular, carbohydrate side chains. In this regard, metabolic labeling withradioactive sugar derivatives or chemical labeling can provide information about thestructure, sequence, and distribution of the sugar chains or glycoconjugates (UNIT 8.13).Treating cells with inhibitors of the enzymes that synthesize N-linked oligosaccharidechains results in production of glycoproteins containing missing or altered chains (UNIT

8.14). This information is useful for ascertaining the presence of such side chains andexamining their functional roles. Further information about the content and nature ofthe oligosaccharide chains present can be obtained by treating the isolated glycoproteinswith various specific glycosidases (UNIT 8.15) and subsequently analyzing the protein inquestion by various techniques (such as SDS-PAGE; UNIT 8.4).

The ultimate proof of the purity of an isolated protein is to demonstrate that it has asingle amino acid sequence. This often can be ascertained by electroeluting the stainedprotein from a polyacrylamide gel (UNIT 8.8) prior to application to a protein sequencer orby directly transferring the protein band to a membrane support, such as PVDF, that iscompatible with automated protein sequencers. Primary sequence analyses can be usednot only to demonstrate purity but to obtain information for synthesizing oligonucleotideprobes that can be used to clone the gene encoding the protein.

Finally, methods for monitoring intracellular phosphorylation-dependent signaling eventson a single-cell basis are included in UNIT 8.17. This approach measures cell signalingby treating cells with exogenous stimuli, fixing cells with formaldehyde, permeabilizingwith methanol, and then staining with phospho-specific antibodies. Thus, cell signalingstates can be determined as a measure of how cells interact with their environment.

It should be kept in mind that many proteins are normally synthesized with the N-terminus group blocked by an acyl moeity and are consequently refractory to Edmandegradation. Therefore, after purification it is often necessary to fragment a protein (bychemical and/or enzymic methods) and to isolate the individual protein fragments byreversed-phase HPLC (Wilson and Schlabach, 1987). The N-terminal sequence analysisof several peptides may then be determined. These data may be used collectively toconfirm the identity of an unknown gene or to form the basis for the synthesis ofoligodeoxynucleotides (primers or probes) or peptides (Chapter 9).

LITERATURE CITED

Borst, J., Alexander, S., Elder, J., and Terhorst, C. 1983. The T3 complex on human T lymphocytes involvesfour structurally distinct glycoproteins. J. Biol. Chem. 258:5135-5141.

Brenner, M.B., McLean, J., Dialynas, D.P., Strominger, J.L., Smith, J.A., Owen, F.L., Seidman, J.G.,Ip, S., Rosen, F., and Krangel, M.S. 1986. Identification of a putative second T cell receptor. Nature322:145-149.

Lew, A.M., Pardoll, D.M., Maloy, W.L., Fowlkes, B.J., Kruisbeek, A.M., Cheng, S.-F., Germain, R.N.,Bluestone, J.A., Schwartz, R.H., and Coligan, J.E. 1986. Characterization of T cell receptor gammachain expression in a subset of murine thymocytes. Science 234:1401-1405.

Weissman, A.M., Samelson, L.E., and Klausner, R.D. 1986. A new subunit of the human T cell antigenreceptor complex. Nature 312:480-482.

Wilson, K.J. and Schlabach, T.D. 1987. Reversed-phase high-performance liquid chromatography. In CurrentProtocols in Molecular Biology (F.M. Ausubel, R. Brent, R.E. Kingston, D.D. Moore, J.G. Seidman,J.A. Smith, and K. Struhl eds.) pp. 10.12.1-10.12.9. John Wiley & Sons, New York.

John E. Coligan