CHAPTER 10 Analysis of Proteinssun-lab.med.nyu.edu/.../CPMB.Ch.10.Analysis.Proteins.pdf(bioinformatics and sequence databases), Chapter 20 (protein-protein interactions), Chapter21(chromatinassemblyandanalysis),

CHAPTER 10Analysis of Proteins

INTRODUCTION

The purification and analysis of proteins isintegral to designing oligonucleotide probesfor gene cloning, confirming DNA sequencedata, and synthesizing peptides for elicitinganti-peptide antibodies. This chapter presentsprotocols for the detection, separation, and pu-rification of proteins. Additional aspects ofprotein analysis are covered elsewhere in thismanual—e.g., in Chapter 12 (DNA-protein in-teractions), Chapter 16 (protein expression),Chapter 17 (protein glycosylation), Chapter18 (protein phosphorylation), Chapter 19(bioinformatics and sequence databases),Chapter 20 (protein-protein interactions),Chapter 21 (chromatin assembly and analysis),Chapter 24 (generation and use of combinato-rial libraries), and Chapter 27 (RNA-proteininteractions).

WHAT IS THE PROTEINPROBLEM?

Briefly put, the protein problem is to tryto understand the diversity, distribution, andfunction of proteins based on specific char-acteristics that can be determined using cur-rent technology. Whether one studies a solitaryprotein (if there is such a reality) or proteincomplexes involved in a biological function,one should ask the following questions: Whatis the molecular mass of the protein being stud-ied? What are the shape and charge of the pro-tein and its various molecular forms? Is theprotein membrane-bound? Does the proteinhave more than one biological function? Whatis the copy number of the protein in the cell?What are the instantiation differences amongcell types and physiological state of the cellsfrom which the protein is being isolated? Is theprotein found in protein complexes? If so, inhow many different complexes? What are thekinetics and thermodynamics of protein com-plex formation? Do the structure and functionof the protein complex depend on cell physi-ology? Is the protein present in more than onecellular compartment? How many molecularforms of the protein are there (i.e., variationsin co- and post-translation, functional degra-

dation products, and complex inhibition oractivation)?

Why is it important to answer these ques-tions? Protein molecular mass varies betweenlow (1,000,000 Da). Aprotein’s molecular mass will affect the choiceof purification method, since chromatographicmatrices and polyacrylamide gels vary in poresize (defining molecular exclusion limits). Theshape of an isolated protein is either globu-lar (usually, an oblate ellipsoid) or fibrillar.Most proteins are globular in their native state(although this generalization may not applyintracellularly for all proteins), and most pu-rification and analysis methods are tailored toworking with globular proteins. The charge ofmost proteins is negative, although some arepositively charged (e.g., histones). This is im-portant to remember when choosing betweenanionic and cationic ion-exchange matrices(in general, choose anionic exchange). Pro-teins are arrays of hydrophilic or hydrophobicamino acids, and subtle changes in the distri-bution of charge and hydrophobicity can leadto changes in biological function and intra-cellular distribution. Proteins can be free ormembrane-bound (i.e., bound to a lipid bi-layer). Free proteins allow the use aqueousbuffers during purification and analysis (oc-casionally requiring addition of mild nonionicdetergents, e.g., for high-molecular-mass pro-teins or protein complexes); membrane-boundproteins always require the use of ionic deter-gents (i.e., SDS) or nonionic detergents (e.g.,Tween, Triton X-100) to keep the protein insolution.

The function of any protein (or protein com-plex) is defined by a specific biological assay.In describing protein function, it is essentialto remember that a protein characterized byone assay may function quite differently inanother, and that an assay might reveal an arti-fact rather than the “true” biological functionof a protein (or protein complex). Further, anassay is generally designed to comply withcurrent biological dogma, and a class-specificprotein assay will only reveal the expected

Contributed by R.K. Scopes and John A. SmithCurrent Protocols in Molecular Biology (2006) 10.0.1-10.0.22Copyright C© 2006 by John Wiley & Sons, Inc.

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Supplement 76

Introduction

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Supplement 76 Current Protocols in Molecular Biology

protein function. The number of protein-coding genes is agreed upon for many organ-isms. However, the number of distinct proteinscontained in any cell and their copy numbershave not been determined unequivocally, sincepurification methods are innately destructiveand detection methods can at best resolve pro-teins present at intermediate (100 to 1000copies per cell) and high (>1000 copies percell) copy numbers. Low-copy-number pro-teins (500,000 discrete proteinswith subtle and possibly duplicative biologi-cal function, depending on their involvementin different pathways. There is no currentlyavailable purification or analysis method thatwould allow the deconvolution of this molec-ular system.

Answering these questions is at the heartof solving the protein problem. This chapterdeals with what is known about protein purifi-cation and characterization. New innovationsin protein science will be required to addressthe questions posed above.

The increasing expansion and sophistica-tion of technologies for protein productionand purification have encouraged the editorsof Current Protocols to continue to expandthe coverage of these issues. Thus, not onlydo several chapters of this manual now dealwith specific aspects of protein analysis, buta companion volume in this series, CurrentProtocols in Protein Science (CPPS; Coliganet al., 2006), is wholly devoted to strategies andprotocols for purification and characterizationof proteins. CPPS provides specific strategiesand recommendations for experimental designof protein purification schemes for both na-tive and recombinant proteins. In addition, itcovers in depth and detail such topics as de-tection and separation of proteins from com-plex mixtures; computational, structural, andfunctional analyses; and detection and analysisof post-translational modifications. Through-out this introduction, reference is made, wher-ever appropriate, to the relevant chapters orunits within Current Protocols in MolecularBiology and CPPS.

OVERVIEW OF PROTEINPURIFICATION ANDCHARACTERIZATION

Aims and ObjectivesMost of the methods used in protein pu-

rification were established by the 1960s and1970s, at least in their principles. More re-cent developments in protein purification havebeen mainly in instrumentation designed tooptimize the application of each methodology.Developments in instrumentation have beenstimulated by the rapid progress in molecu-lar biology, because gene isolation has oftenbeen preceded by isolation of the gene prod-uct. Because such products can now be charac-terized sufficiently (i.e., partially sequenced)

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Current Protocols in Molecular Biology Supplement 76

using minute amounts of protein, the need forlarge-scale or even moderate-scale procedureshas decreased. Hence there has been an ex-plosive development of modern equipment de-signed specifically for dealing with amounts ofprotein in the milligram to microgram range.On the other hand, structural studies usingX-ray crystallography and nuclear mag-netic resonance (NMR) require hundreds ofmilligrams of pure protein, so larger-scaleequipment and procedures are still needed inthe research laboratory.

As more proteins, and particularly en-zymes, were purified and crystallized, theystarted to be used increasingly in diagnostic as-says and enzymatic analyses, as well as in thelarge-scale food, tanning, and detergent indus-tries. Many enzymes used in industry are notin fact very pure, but as long as they do the jobneeded, that is sufficient. “Process” enzymessuch as α-amylase, proteases, and lipases areproduced in ton quantities, mainly as secretionproducts in bacterial cultures, and to minimizecosts may undergo only limited purificationprocesses. At the other extreme, enzyme prod-ucts for research and analysis require a highdegree of purification to ensure that contam-inating activities do not interfere with the in-tended use. Anyone familiar with molecularbiology enzymes will appreciate how minutelevels of contamination with DNase or RNasecan completely destroy carefully plannedexperiments.

The nature of the proteins studied has alsochanged substantially. Whereas enzymes wereonce the most favored subjects for research,they have now been superceded by nonen-zymatic proteins such as growth factors, hor-mone receptors, viral antigens, and membranetransporters. Many of these are present inminute amounts in their natural source, andtheir purification can be a major task. Heroicefforts in the past have used kilogram quan-tities of rather unpleasant starting materials,such as human organs, and ended up with afew micrograms of pure product. It is nowmore usual, however, to take the genetic ap-proach: clone the gene before the protein hasbeen isolated or even properly identified, thenexpress it in a suitable host cell culture or or-ganism. The expression level may be orders ofmagnitude higher than in the original source,which will make purification a relatively sim-ple task. It can be useful to know beforehandsome physical properties of the protein, to fa-cilitate the development of a suitable purifica-tion protocol from the recombinant source. Onthe other hand, there are now several ways of

preparing fusion proteins, which can be puri-fied by affinity techniques without any knowl-edge of the properties of the target protein (seeChapter 16). Moreover, there are ways of mod-ifying the expressed product to simplify purifi-cation further (UNITS 10.11B & 20.2).

Thus, the approach to protein purificationmust first take into account the reason it is be-ing done, as the methods will vary greatly withdifferent requirements. At one extreme is theone-of-a-kind purification, in a well-financedand equipped laboratory, that is carried out toobtain a small amount of product for sequenc-ing so that gene isolation can proceed. Here,the expense of equipment and reagents maypose no problem, and a very low overall recov-ery of product can be acceptable, provided theproduct is pure enough. At the other extremeare the requirements of commercial produc-tion of a protein in large amounts on a contin-uing basis, where high recovery and economyof processing are the chief parameters to beconsidered. There are many intermediate situ-ations as well.

Many publications in the area of protein re-search are entitled “Purification and character-ization of. . .,” and describe a purification pro-cedure in sufficient detail that it can be repro-duced in another laboratory. The characteriza-tion section may include structural, functional,and genetic information, and carrying out suchstudies is likely to require at least milligramquantities of pure protein. Ideally the purifica-tion should involve a small number of steps,with good recovery at each step. If the recov-ery is poor (

Introduction

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but may be overlooked at first, and that canhave a major effect on the purification proce-dure even if varied only slightly. The processbeing reported should always be repeated ex-actly as described before a manuscript is sub-mitted for publication. There is one exception,namely, the case where purification was con-ducted simply to obtain enough protein forsequencing and gene isolation; if those objec-tives were achieved, there should be no needto provide instructions for repetition.

Sources of Material for ProteinPurification

For many people embarking on a proteinpurification project, there is no choice of ma-terial. They are studying a particular biolog-ical tissue or organism, and the objective isto purify a protein from that source. However,there may be approaches that can make theproject simpler. If, for instance, the source isdifficult to obtain in large amounts, it may bebest to carry out at least preliminary trials ona source species more readily obtained. Themost obvious and relevant example is whenthe species being studied is Homo sapiens,and tissue samples are not readily availablefor practical or ethical reasons, or both. Inthis case, it is usual to go to where mam-malian tissue is readily available (i.e., an abat-toir) and work with bovine, ovine, or porcinesources. Alternatively, if quantity of tissue isnot a problem, the humble laboratory rat maysuffice. Once a protocol for purifying the pro-tein from substitute sources has been workedout, it will be much easier to develop one us-ing human material—the identical proceduremay work satisfactorily. Proteins differ to afairly small extent between species that havediverged within ∼100 million years, a timeframe that groups together most higher mam-mals. Thus, the behavior of proteins derivedfrom different animals with respect to the var-ious fractionation procedures is likely to besimilar, and a protocol worked out for pig tis-sues is likely to need only minor adjustmentsfor application to human tissues.

A second example is where the interestis mainly in the function of a protein, espe-cially an enzyme, for which functions and ac-tions have generally been strongly conservedthrough evolution. In that case, a preliminaryscreening of potential sources or, better still,the literature should provide a raw materialthat is best suited to the investigator’s pur-poses. Considerations should include the fol-lowing: (1) What functions are required of theend product? For instance, an enzyme having a

low Km may be needed, so selecting the sourcewith the highest activity may not suffice.(2) How convenient is it to grow or obtain theraw material, and are there problems relatingto pathogenicity or extractability? (3) Does thequantity of the protein vary with growth con-ditions or age, and does the protein deterioratein situ if left too long? Obviously one requiresa source that reliably produces the highestamount of the desired protein per unit volumeto maximize the chances of developing a goodpurification procedure. (4) What storage con-ditions are required for the raw material? It isimportant to consider that fresh raw materialmay not be immediately available whenever apurification is attempted.

The above considerations are relevant to thetraditional situation for commencing a proteinpurification project. It is becoming increas-ingly common, however, for proteins to bepurified as recombinant products using tech-niques in which the gene is expressed in a hostorganism or in cultured cells. This of courserequires that the gene encoding the protein ofinterest be available. Until the mid-1980s, suchmaterial was usually obtained by hybridizationof an oligonucleotide synthesized accordingto amino acid sequence information. This re-quired the protein to have been purified first,so the initial task of protein purification stillneeded to be done at least once. More recently,genetic techniques have permitted the isola-tion of many genes encoding known proteins,even though the proteins may never have beenstudied directly. Moreover, with the comple-tion of the Human Genome Project and relatedDNA sequencing efforts, many genes for bothknown and unknown proteins have becomeavailable and can be expressed in recombinantform without ever being purified from the hostspecies. As a result some completely new con-siderations for protein purification come intoplay, including the possibility of modifyingthe gene structure not only to increase expres-sion level and alter the protein product itselfto enhance a desired function, but, equally im-portantly, to aid in purification. Recombinantproteins may be expressed in bacteria, yeasts,insect cells, and animal tissue cultures. Furtherdetails may be found in Chapter 16.

Detection and Assay of ProteinsDuring a protein purification procedure

there are two measurements that need to bemade, preferably for each fraction: total pro-tein and amount of the desired protein (usu-ally assessed in terms of bioactivity). Detailsof the most commonly used assay methods are

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given in UNIT 10.1A. It is not possible to iso-late a protein without a method of determiningwhether it is present; an assay, either quan-titative or at least semiquantitative, indicatingwhich fraction contains the most of the desiredprotein is essential.

Assays may range from the quick-and-easytype (e.g., instantaneous spectrophotometricmeasurement of enzyme activity) to long andtedious bioassays that may take days to pro-duce an answer. The latter situation is very dif-ficult, because by the time one knows wherethe protein is, it may be “was,” owing to degra-dation or inactivation. Moreover, this may notbecome clear until the next step has been com-pleted and its products assayed. Any assay thatis quick is therefore advantageous, even if thismeans sacrificing accuracy for speed.

Measurement of total protein is useful, asit indicates the degree of purification at eachstep. However, unless the next step criticallydepends on how much protein is present, mea-surement of total protein is not extremely im-portant: a small sample can be put aside andmeasured later, when the purification is com-plete. It is, however, very important to knowhow much protein is present in the final, pre-sumed pure sample, as this will indicate thespecific activity (if the protein has an activ-ity), which can be compared with that of otherpreparations. The general object is to obtainas high a specific activity as possible (takinginto account recovery considerations), whichmeans retaining as much of the desired proteinas possible while ending up with as little totalprotein as possible.

Methods for Separation andPurification of Proteins

The methods available for protein purifi-cation range from simple precipitation pro-cedures used since the nineteenth century tosophisticated chromatographic and affinitytechniques that are constantly undergoing de-velopment and improvement. Methods can beclassified in several alternative ways; perhapsone of the best is based on the properties ofthe proteins that are being exploited. Thus, themethods can be divided into four distinct butinterrelated groups depending on protein char-acteristics: surface features, size and shape, netcharge, and bioproperties.

Methods based on surface featuresSurface features include charge distribution

and accessibility, surface distribution of hy-drophobic amino acid side chains, and, to alesser extent, net charge at a given pH (see

discussion of net charge, below). Methods ex-ploiting surface features depend mainly onsolubility properties. Differences in solubil-ity result in precipitation by various manip-ulations of the solvent in which the proteinsare solubilized. Methods for obtaining an ex-tract containing the desired protein in solubleform are given in Chapter 4 of CPPS. Thesolvent, nearly always water containing a lowconcentration of buffer salts, can be treated toalter properties such as ionic strength, dielec-tric constant, pH, temperature, and detergentcontent, any of which may selectively precip-itate some of the proteins present. Conversely,proteins may be selectively solubilized froman insoluble state by manipulation of the sol-vent composition. The surface distribution ofhydrophobic residues is an important deter-minant of solubility properties; it is also ex-ploited in hydrophobic chromatography, bothin the reversed-phase mode (UNITS 10.12-10.14)and in aqueous-phase hydrophobic-interactionchromatography.

Also included in this category is the highlyspecific technique of immunoaffinity chro-matography (UNIT 10.11A), in which an anti-body directed against an epitope on the proteinsurface is used to pull out the desired proteinfrom a mixture.

Methods based on whole structure:Size and shape

Although the size and shape of proteins canhave some influence on solubility properties,the chief method of exploiting these propertiesis gel-filtration chromatography (UNIT 10.9).In addition, preparative gel electrophoresismakes use of differences in molecular size.Proteins range in size from the smallest clas-sified as proteins rather than polypeptides(∼5000 Da) up to macromolecular complexesof many million daltons. Many proteins in theirbioactive states are oligomers of more thanone polypeptide, and these can be dissociated,though normally with loss of overall struc-ture. Thus, many proteins have two “sizes”:that of the native state and that (or those)of the polypeptides in the denatured and dis-sociated state. Gel-filtration procedures nor-mally deal only with native proteins, whereaselectrophoretic procedures commonly in-volve separation of dissociated and denaturedpolypeptides.

Methods based on net chargeTwo separation techniques exploit the over-

all charge of proteins: ion-exchange chro-matography (by far the most important) and

Introduction

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electrophoresis (UNITS 10.2A, 10.3 & 10.4; see alsoChapter 10 of CPPS). Ion exchangers bindcharged molecules, and there are essentiallyonly two types of ion exchangers, anion andcation. The net charge of a protein depends onthe pH—positive at very low pH, negative athigh pH, and zero at some specific point inbetween, termed the isoelectric point (pI). Itshould be stressed that at its pI a protein hasa great many charges; it just happens that atthis pH the total negatives exactly equals thetotal positives. The most charged state (dis-regarding the charge sign) is in the pH range6.0 to 9.0. This is the most stable pH rangefor most proteins, as it encompasses commonphysiological pH values. Ion exchangers con-sist of immobilized charged groups and attractoppositely charged proteins. They provide themode of separation that has the highest reso-lution for native proteins. High-performancereversed-phase chromatography has equiva-lent or even better resolution, but it generallyinvolves at least partial denaturation during ad-sorption and is not recommended for sensitiveproteins such as enzymes. Protein purifica-tion using ion-exchange chromatography hasmainly employed positively charged anion ex-changers, for the simple reason that the major-ity of proteins are negatively charged at neutralpH (i.e., have a low isoelectric point). Detailsof this methodology are found in UNIT 10.10.

Methods based on bioproperties(affinity)

A powerful approach for separating the de-sired protein from others is to use a biospe-cific method in which the particular biologi-cal property of the protein is exploited. Onesuch property is the affinity between a pro-tein’s binding site and its ligand: the ligand,when immobilized, attracts the protein froma mixture, while other molecules are washedaway (UNITS 10.11B & 24.5). This general ap-proach is known as affinity chromatography;there are also nonchromatographic methods ofexploiting the same concept (UNITS 10.20, 10.21,10.24 & 10.27). Although this direct affinity islimited to proteins with a natural binding ac-tivity, most proteins of interest do have specificligands: enzymes have substrates and cofac-tors, and hormone-binding proteins, growthfactors, and receptor molecules are designedto bind specifically and tightly to their naturalpartners. There are also other related affinitymethods for nonbinding proteins. One is im-munoaffinity chromatography (UNIT 10.11A), inwhich the “ligand” is an antibody to the pro-tein in question. An equally important and

now widely used method is the creation ofa fusion protein whose fused portion has astrong affinity toward an immobilized ligand(Chapter 16). For this latter technique it is notnecessary for the protein of interest to have anybinding property of its own; the fusion portionpossesses a strong affinity for standardized im-mobilized ligands.

Characterization of the ProteinProduct

Once a pure protein is obtained, it may beemployed for a specific purpose such as enzy-matic analysis (e.g., glucose oxidase and lac-tate dehydrogenase) or as a therapeutic agent(e.g., insulin and growth hormone). However,it is normal, when a protein has been isolatedfor the first time, to characterize it in termsof structure and function. Several features aregenerally expected to be addressed (and re-ported in the literature) in the characterizationof a new protein. One is the molecular weight,or at least the size of the subunit(s), whichis determined by SDS-PAGE (UNITS 10.2A-10.5)and/or gel filtration (UNIT 10.9). Spectral prop-erties such as the UV spectrum (Trp and Tyrcontent) and circular dichroism (CD) spec-trum (secondary structure), and special char-acteristics of proteins with prosthetic groups(e.g., quantitation and spectra), may also bedescribed. For glycoproteins, the number andnature of substituent carbohydrates should bedetermined (Chapter 17). Furthermore, if thegene has not already been reported, someamino-terminal sequence analysis should begiven, if at all possible, along with the re-sults of a database search for similar se-quences (UNIT 19.3). Functional proteins shouldbe demonstrated to have the appropriate func-tion, and detailed kinetic characterization isappropriate for enzymes. Ultimately the fullthree-dimensional structure of the protein maybe determined, which will require crystals; anysuccessful crystallization attempts should bereported.

The Protein Purification LaboratoryThe requirements for a protein purification

laboratory cannot be exactly formulated be-cause they depend greatly on the types andamounts of proteins being isolated. To cover alleventualities, it would be necessary to have oneset of equipment to deal with submicrogramquantities and another set to deal with multi-gram quantities—a range of ∼108! One lab-oratory dedicated to protein purification maynot need small-scale equipment if, for exam-ple, its work involves plasma proteins that are

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always available in large quantities. Anothermay have all the latest in high-performanceequipment but not be able (nor need) to han-dle quantities of protein in excess of a fewmilligrams.

If it is assumed that neither extreme in quan-tity is to be attempted, and that the labora-tory is handling a variety of protein types andsources, then certain basic pieces of equipmentare needed. Obtaining the starting material andmaking an extract of it require homogenizationequipment and centrifuges to remove insolu-ble residues. Preliminary fractionation start-ing from a crude extract of tissue or cells re-quires equipment and materials that will notbecome clogged by particulates. Adsorbentsand similar materials used at the first stepshould be relatively inexpensive so that whenperformance falls off after a few uses, owing tointransigent impurity buildup, they can be dis-carded. It is also relevant that a larger amountis handled at the initial step than later steps;therefore, reagent expense can be an impor-tant consideration. After the first one or twosteps, the sample should be sufficiently cleanand clear to permit use of high-performanceequipment.

High-performance liquid chromatography(HPLC) is a term with a variety of meanings(see UNITS 10.12-10.14). To some it refers exclu-sively to reversed-phase chromatography (heredistinguished as RP-HPLC); to others it in-cludes all sorts of chromatography, providedthat the equipment is fully automated and high-performance adsorbents are used. A high-performance system designed specifically forproteins—produced by Amersham PharmaciaBiotech (see APPENDIX 4) under the name FastProtein Liquid Chromatography or FPLC—uses standard protein chromatographies suchas ion exchange, hydrophobic interaction, andgel filtration. Scaleup is possible with largerequipment based on the FPLC design, so thatlaboratory development can be quickly trans-lated to large-scale production. FPLC is de-signed to separate proteins in their native ac-tive configuration, whereas RP-HPLC oftencauses at least transient denaturation duringadsorption and elution. RP-HPLC has a highresolving power, but it is best suited to peptidesand proteins smaller than ∼30 kDa. Chro-matography run with older-style low-pressureadsorbents is sometimes referred to as “low-performance” or “open-column” chromatog-raphy; neither of those descriptions is neces-sarily accurate. These methods require simplefraction collector and monitoring equipment.This equipment will be used for larger-scale

operations (tens of milligrams of protein andupward), probably at an earlier stage in theprotocol than with HPLC.

Various columns, both prepacked withproprietary adsorbents and empty for self-packing, will be needed, with the sizes andtypes depending on the scale of operations.Several anion-exchange columns of differentsizes, one or two cation-exchange columns,and gel-filtration media are all essential, alongwith a range of alternative adsorbents such ashydrophobic interaction materials, dyes, hy-droxyapatite, and chromatofocusing and spe-cialist affinity media.

Fully equipped protein purification lab-oratories should also have preparativeelectrophoresis and isoelectric focusing ap-paratuses for the rare occasions when othertechniques fail to give sufficient separation.

In addition to equipment used in the ac-tual fractionation processes, a variety of otheritems are needed. In particular it is importantto be able to change buffers quickly and toconcentrate protein solutions with ease. Theseoperations require such items as dialysis mem-branes (UNIT 10.5 & APPENDIX 3C), ultrafil-tration cells (APPENDIX 3C), and gel-filtrationcolumns of various sizes (UNIT 10.9).

Finally, equipment for assaying and ana-lyzing the preparations is needed. Most suchequipment is fairly standard in biochemicallaboratories and includes spectrophotometers,scintillation counters, analytical gel and capil-lary electrophoresis apparatuses, immunoblot-ting materials, and immunochemical reagents.A listing of standard equipment is found inAPPENDIX 2.

STRATEGIES FOR PROTEINPURIFICATION

Classification of ProteinsAs with most heterogeneous collections

of things, proteins can be classified in sev-eral different ways, such as by function, bystructure, or by physicochemical characteris-tics. Each protein species consists of identicalmolecules with exactly the same size, aminoacid sequence, and three-dimensional shape.In this way a solution of a mixture of pro-teins differs from a solution of synthetic poly-mers or sheared DNA, both of which contain acomplete spectrum of possible sizes centeredaround the average. The protein mixture hasonly discrete sizes of molecules correspond-ing to each type of protein present. Althoughit would be possible to classify proteins bysize, it would be of limited use, as there is

Introduction

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Table 10.0.1 Classification of Proteins by Structural Characteristics

Structuralcharacteristic

Examples Comments

Monomeric Lysozyme, growth hormone Usually extracellular; often havedisulfide bonds

Oligomeric

Identical subunits Glyceraldehyde-3-phosphatedehydrogenase, catalase, alcoholdehydrogenase, hexokinase

Mostly intracellular enzymes; rarelyhave disulfide bonds

Mixed subunits Aspartate carbamoyltransferase,pertussis toxin

Allosteric enzymes; differentsubunits have separate functions

Membrane-bound

Peripheral Mitochondrial ATPase, alkalinephosphatase

Readily solubilized by detergents

Integral Porins, cytochromes P450, insulinreceptor

Require lipid for stability

Conjugated Glycoproteins, lipoproteins,nucleoproteins

Many extracellular proteins containcarbohydrate

Table 10.0.2 Classification of Proteins by Function

Function Examples

Amino acid storage Seed proteins (e.g., gluten), milk proteins (e.g., casein)

Structural

Inert Collagen, keratin

With activity Actin, myosin, tubulin

Binding

Soluble Albumin, hemoglobin, hormones

Insoluble Surface receptors (e.g., insulin receptor), antigens (e.g., viral coatproteins)

With activity Enzymes, membrane transporters (e.g., amino acid uptake systems,ion pumps)

usually no obvious relationship between sizeand function.

A more useful structural classification takesinto consideration shape and oligomeric struc-ture (Table 10.0.1). In part, structure reflectsbiological location and origin. Simple, fairlyrigid protein molecules occur in the extracellu-lar environment, more complex and readily de-activated molecules are found intracellularly,and hydrophobic proteins are associated withmembranes.

Classification by function is even more rel-evant (Table 10.0.2). Proteins can be simplystores of amino acids, can be structural, orcan have specific binding functions. The most

“functional” proteins are enzymes, which haveboth binding and catalytic roles. In part, thisreflects the degree to which the detailed struc-ture is a requirement for the protein’s func-tion, which in turn relates to conservation ofstructure through evolution. But as with everyattempt at classification, there will always beexamples that do not fit the pattern well. Mostproteins of interest to the pharmaceutical in-dustry belong to the general class of bindingproteins: for instance, hormones (e.g., insulinand bovine somatostatin), viral antigens (e.g.,hepatitis B antigen), growth factors [e.g., in-terferons, interleukins and colony-stimulatingfactors (CSFs)], and antibodies.

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Strategies Based on Classification

Soluble extracellular proteinsThe source of soluble extracellular proteins

is the extracellular medium, whether it be ananimal source such as blood or spinal fluid,or a culture medium in which bacterial, fun-gal, animal, or plant cultures have been grown.Generally these do not contain a large numberof different proteins (blood is an exception),and the desired protein may be a major com-ponent, especially if produced as the result ofrecombinant expression. Nonetheless, the pro-tein in the starting material may be quite dilute,and a large volume may therefore need to beprocessed. The starting fluid may also containmany compounds other than proteins, whosebehavior must be taken into account. The firststage should aim mainly to reduce the vol-ume and remove as much nonprotein materialas possible; some protein-protein separation isalso useful, but not essential. No general rulescan be given, but a batch adsorption method us-ing an inexpensive material such as hydroxya-patite, ion-exchange resin, immobilized metalaffinity chromatography (IMAC) medium, oraffinity adsorbent is best, if feasible. Followingthe first step, the sample should be in a formthat is amenable to standard purification pro-cesses such as precipitation and column chro-matography (see Chapter 8 of CPPS).

Intracellular (cytoplasmic) proteinsTo obtain soluble intracellular proteins

(which are mainly enzymes), cells must be bro-ken open or lysed to release their soluble con-tents. The ease with which cell disruption canbe accomplished varies considerably; animalcells are readily broken, as are many bacte-ria, but plants and fungi have tough cell walls.Methods for obtaining cell extracts are given inChapter 4 of CPPS. The macromolecular sol-uble contents of cells are mainly proteins, withnucleic acids as a minor but significant compo-nent. Bacterial extracts may be viscous unlessDNase is added to break down the long DNAmolecules. Although chromatographic proce-dures can be applied to crude extracts, valuablehigh-performance materials should not be em-ployed in the first step, as there are alwayscompounds, including unstable proteins, thatmay bind to them and be difficult to remove.

Membrane-associated proteinsThere are two approaches to isolating a

membrane-associated protein. In one method,the relevant membrane fraction can first beprepared and then used to isolate the protein.Alternatively, whole tissue can be subjected to

an extraction that solubilizes the membranesand releases the cytoplasmic contents as well.The former approach is much better in thatpurification is accomplished by isolating themembranes: the specific activity of the solubi-lized membrane fraction will be much higherthan with the second method. However, theprocess of purifying the membrane fractionmay lead to substantial losses, and it may bedifficult to scale up. If total recovery of theprotein is more important than purity, a whole-tissue extract is likely to be more appropriate.Although this means that a greater degree ofpurification is needed, the fact that membraneproteins, by definition, have properties some-what different from those of cytoplasmic pro-teins permits some very effective purificationsteps (e.g., hydrophobic chromatography).

Peripheral membrane proteins are onlyloosely attached and may be released by gen-tle conditions such as high pH, EDTA, or low(nonionic) detergent concentrations. Once insolution, some peripheral proteins no longerrequire the presence of detergent to maintaintheir solubility. Integral membrane proteinsare much more demanding—they require highconcentrations of detergent for solubilization(i.e., complete solubilization of the membraneis needed to release them) and generally areneither soluble nor stable in the absence ofdetergent. It is sometimes necessary to main-tain natural phospholipids in association withthe proteins in order to maintain activity. Evenwhen the final objective does not require ac-tivity (e.g., protein sequencing), it is generallynecessary to maintain bioactivity so that anassay can be used to monitor the presence ofthe protein during the purification process. Ifa particular band on a gel is known to be thedesired protein, then no other assay is neededand loss of bioactivity can be allowed.

Purification processes may be affected bythe presence of detergents. The problem of as-sociation with detergent micelles makes puri-fying integral membrane proteins difficult; theclose association of the different proteins orig-inating from membranes often results in verypoor separation in conventional fractionationprocedures.

Insoluble proteinsNatural proteins that are insoluble in nor-

mal solvents are generally structural proteins,which are sometimes cross-linked by post-translational modification. The first stage ofpurification is obvious—it involves extractingand washing away all proteins that are soluble,leaving the residue containing the desired

Introduction

10.0.10


material. Further purification in a native state,however, may be impossible; extracting awayother proteins using more vigorous solvents orattempting to solubilize the target protein maydestroy the natural structure. Cross-linked pro-teins such as elastin or collagen cannot be dis-solved without breaking the cross-links, andthe individual proteins may even be cross-linked together.

Insoluble recombinant proteins(inclusion bodies)

A major new class of insoluble proteinsare recombinant proteins expressed (usually inE. coli) as inclusion bodies. These are denseaggregates found inside cells that consistmainly of a desired recombinant product, butin a nonnative state. Inclusion bodies may formfor a variety of reasons, such as insolubility ofthe product at the concentrations being pro-duced, inability to fold correctly in the bacte-rial environment, or inability to form correct,or any, disulfide bonds in the reducing intracel-lular environment. Their purification is simple,as the inclusion bodies can be separated bydifferential centrifugation from other cellularconstituents, giving almost pure product; theproblem is that the protein is not in a nativestate and is insoluble. Some methods for ob-taining an active product from inclusion bodiesare described in CPPS (Palmer and Wingfield,2004).

Soluble recombinant proteinsRecombinant proteins that are not ex-

pressed in inclusion bodies either will be sol-uble inside the cell or, if an excretion vector isbeing used, will be extracellular (or, if E. coliis the host, possibly periplasmic). They canbe purified by conventional means. In somesystems, expression is so good that the de-sired product is the major protein present andits purification is relatively simple. In systemswhere the expression level is low, the purifica-tion process can be tedious—though easier, itcan be hoped, than isolation from the naturalsource. It should be remembered that a proce-dure developed for isolating a protein from nat-ural sources may not work successfully withthe recombinant product, because the natureof the other proteins present influences manyfractionation procedures.

Many recombinant proteins are now pro-duced in a fusion form in which the fusionportion is designed to facilitate purificationby affinity chromatography (UNIT 10.11B). Avariety of “fusion tags” are available as partof the expression vector (see UNITS 16.6–16.8

and Sassenfeld, 1990, for specific examples).These include whole proteins such as pro-tein A, glutathione-S-transferase, and maltose-binding protein, as well as short peptide seg-ments that may be recognized by a specificantibody, become biotinylated in the host cell,or bind tightly to an immobilized metal ionadsorbent. Nearly complete purification canbe achieved in one step by passing the crudeextract through an appropriate adsorbent andeluting specifically. A further advantage of thefusion approach is that expression levels arelikely to be determined more by the transcrip-tion and translation signals for the fusion pep-tide (which have been optimized in the vector)than by the structural features of the insertedgene. The only remaining task is to removethe fusion peptide, though for some purposesthis is not necessary (see UNIT 16.4B). This re-quires a proteolytic step: highly specific pro-teases such as blood clotting factors are gener-ally used, their recognition sites having beenincorporated in the sequence joining the fusionpeptide. It is sometimes convenient to “digest”the fusion protein from the adsorbent using theprotease.

Unstable proteins may be modified by themolecular biological technique of site-directedmutagenesis to remove the site of instability—for instance, an oxidizable cysteine. Such tech-niques are appropriate for commercial pro-duction of proteins, but may of course alternatural functioning parameters. One modifi-cation that can be useful is increased ther-mostability, although it is not easy to predictmutations that will improve that parameter.Thermostable proteins originating from ther-mophilic bacteria do not need structural modi-fication and, if expressed in large amounts, canbe purified satisfactorily in one step by simplyheat-treating the extract at 70◦C for 30 min,which will denature virtually all the host pro-teins (e.g., see Oka et al., 1989).

The host bacteria used for production ofrecombinant proteins are usually E. coli orBacillus subtilis; they may express proteinsat anywhere from 1% to >50% of the cellu-lar protein, depending on such variables as thesource, promoter structure, and vector type.Generally the proteins are expressed intracel-lularly, but leader sequences for excretion maybe included. In the latter case, the protein isgenerally excreted into the periplasmic space,which limits the amount that can be produced.Excretion from gram-positive species such asB. subtilis sends the product into the culturemedium, with little feedback limitation on thetotal expression level.

Analysis ofProteins

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PROTEIN PURIFICATION FLOWCHARTS

Protein purification flow charts are pre-sented to give a broad outline of the methodsused for different types of proteins. They can-not give any detail, as the process appropriatefor each protein will have its own variations ateach stage. In most cases, the first stage is toobtain a solution containing the desired pro-tein, after which it can be dealt with by themany separation techniques described in thefollowing chapters. In some cases the insolu-bility of the desired protein can be exploited byremoving soluble fractions. Purification proce-dures are commonly divided into three stages:(1) the primary steps deal with crude mix-tures of proteins and other molecules presentin the raw material; (2) the secondary process-ing generates a product near homogeneity; and(3) the polishing steps remove minor contam-inants, a process that is especially importantfor therapeutic proteins.

Soluble Recombinant ProteinsProteins expressed in a recombinant man-

ner may be (1) soluble in the cytoplasm,(2) insoluble as inclusion bodies, (3) excretedfrom the cells into the culture medium,(4) excreted into the periplasmic space (e.g.,in gram-negative bacteria), or (5) associatedwith organelles or membrane fractions. Inaddition they may be expressed (6) as thenormal, mature, naturally occurring protein,(7) containing a natural leader peptide thatwould normally be processed, (8) as a fusionprotein with a peptide that is not natural to theprotein, or (9) lacking glycosylation or otherposttranslational modification, or modified in-correctly. Possibilities (1) to (5) affect themethod of extraction used to obtain the startingmaterial for purification. Cases (6) to (9) canaffect the methods used for purification.

The scheme for purifying soluble recom-binant proteins is outlined in Figure 10.0.1.The first stage is to obtain a clarified solutioncontaining the desired protein, with as little inthe way of unwanted proteins as possible. Forsoluble cytoplasmic proteins, case (1), it is notnormally possible to exclude any significantamount of unwanted soluble proteins, but incases (2) to (5) the compartmentalization awayfrom the cytoplasm allows such separation inthe initial stage.

It may be necessary to carry out a con-centration step before proceeding, especiallyif the protein has been excreted into theculture medium. Normally ultrafiltration isused (see APPENDIX 3C), although other tech-

niques are possible, especially if the extractcontains particulates that block ultrafiltrationmembranes.

Recombinant expression in the cytoplasmof bacteria followed by extraction via totalcell disruption results in large amounts of nu-cleic acids being solubilized with the protein.A number of treatments to remove nucleicacids are possible. Streptomycin is used to pre-cipitate ribosomal material, and cationic poly-mers such as protamine (a basic protein) andpolyethylenimine will form insoluble com-plexes (at low ionic strength) with nucleicacids. In addition, viscosity caused by DNAcan be reduced by adding small amounts ofDNase.

Insoluble Recombinant ProteinsIt has been found that many proteins do

not fold correctly when expressed in bacteria(mainly in E. coli), and as a result aggrega-tion occurs, leading to large insoluble inclu-sion bodies within the cytoplasm of the cells(see Chapter 6 of CPPS). Although this cre-ates major difficulties in obtaining satisfactoryamounts of active native product, it greatlysimplifies the initial stage of purification.

The purification scheme for recombinantinsoluble proteins is outlined in Figure 10.0.2.After cell disruption, inclusion bodies can beobtained in a fairly pure state by differentialcentrifugation. They must then be solubilized,however, and the active protein generatedby encouraging correct folding. Solubilizationis usually accomplished with guanidine hy-drochloride and/or urea, and thiols such as 2-mercaptoethanol or glutathione are includedto disrupt any disulfides that have formed andprevent more from forming. Folding the pro-tein correctly may require a variety of addi-tions to the solution, as well as slow removalof the denaturant. The latter can be car-ried out by simple dilution or by dialysis(APPENDIX 3C). Folding occurs best at lowprotein concentrations, so dilution may beadequate. If the native protein does containdisulfides, then it is important to create redoxconditions such that some (but not excessive)oxidation of thiols can occur. A combinationof oxidized and reduced glutathione is com-monly used. In addition, the action of the en-zyme protein disulfide isomerase, which canmake and unmake disulfides by exchange re-actions, has been found to be beneficial inmany cases. If the native protein is of intracel-lular origin, it probably will not contain disul-fides; it will, however, contain cysteines, so afull reducing potential should be maintained.

Introduction

10.0.12


Figure 10.0.1 Purification scheme for soluble recombinant proteins, which may be excretedor located in the periplasm, the membrane fraction, or most commonly the cytoplasm. The firststep is to obtain an extract containing the desired protein in soluble form. After this, conventionalpurification steps may be carried out, or affinity purification of tagged fused proteins can beperformed.

Specific methodology is discussed in CPPS(Wingfield, 1995; Wingfield et. al., 1995).

Not all proteins can fold unassisted by othercellular components. Chaperonins are proteinswhose role is to assist in folding proteins in-cluding those unfolded by heat shock (Zeilstra-Ryalls et al., 1991). The ones most studied,which are just becoming commercially avail-able as of 1996, are the E. coli chaperoninsGroEL and GroES, both of which are needed,together with ATP, to renature many proteins.Proline residues can adopt two isomeric con-formations in proteins, and the wrong con-

formation is switched to the correct one bythe enzyme prolyl isomerase, aiding the pro-cess of protein folding. At present these arenot large-scale prospects, both because ofthe cost of the chaperonins and because theagents operate best in vitro at very low proteinconcentrations.

Once the proteins are folded, the purifi-cation process consists of removing smallamounts of still incorrectly folded protein plusany other host proteins that were trapped withthe original inclusion bodies. The former maybe difficult, as incorrectly folded species have

Analysis ofProteins

10.0.13


Figure 10.0.2 Purification scheme for insoluble recombinant proteins that are produced as in-clusion bodies in the cytoplasm of host cells. The cells must be broken open and the insolubleinclusion bodies separated by differential centrifugation. Solubilization is achieved by the use ofdenaturing solvents, and renaturation of the dissolved protein occurs on removal of the denatu-rant. Further polishing steps will be needed to remove small amounts of contaminating proteins aswell as incorrectly folded species. Additional information can be found in CPPS (see Pain, 1995;Wingfield et al., 1995; Palmer and Wingfield, 2004).

a size and charge similar to those of the correctproduct. However, subtle differences arisingfrom the folded conformation can be exploitedby chromatographic techniques. In ideal casesimmunoaffinity techniques using antibodiesspecific for either the incorrectly folded formor the correct one can be used to resolve themixture.

Soluble Nonrecombinant ProteinsThere are so many sources of soluble pro-

teins that it is not possible to give a completeoverview of methods used to obtain startingextracts from which a desired protein can beisolated. The sources can be classified as eithermicroorganisms, plants, or animals, as shownin Figure 10.0.3, but these in turn should besubdivided according to how the starting ex-

tract is obtained. In particular there is a dis-tinction between extracellular and intracellu-lar proteins. With the latter it is necessaryto disrupt the cells and release the proteins,whereas with the former, if the extracellularfluid can be obtained directly, there need beno contamination with intracellular proteins.Extracellular sources include microorganismculture medium, plant and animal tissue cul-ture medium, venoms, milk, blood, and cere-brospinal fluids. Soluble proteins may also oc-cur within organelles such as mitochondria;these may best be obtained by first isolatingthe organelle, then disrupting it to release thecontents.

The starting extract normally contains be-tween 5 and 20 mg protein per milliliter,though lesser concentrations can be dealt with,

Introduction

10.0.14


Figure 10.0.3 Purification scheme for soluble proteins present in their natural host cells. Cellsmust be disrupted to release the proteins, usually in the presence of 2 to 10 ml of a suitable bufferper gram weight. After removal of insoluble material, the process will generally require severalsteps, using various standard fractionation procedures in a suitable order. For production of highlypure protein, a final polishing step may be required to remove final trace contaminants. Additionalinformation can be found in CPPS (see Wingfield, 2005).

especially if working on a small scale. It maybe necessary to include a concentration stepbefore starting the purification process in or-der to approach that level. There are exceptionsto every rule, however, and very high pro-tein concentrations can be handled, for exam-ple, with two-phase partitioning (Walter andJohansson, 1994). When isolating proteins on

a large scale, the volumes being manipulatedbecome of increasing concern, so maximizingprotein concentration can be an important aim.The starting extract should be clarified, usu-ally by centrifugation; on a large scale, ultra-filtration methods (APPENDIX 3C) are becomingmore widely used. Pretreatment of certain ex-tracts to remove excessive amounts of nucleic

Analysis ofProteins

10.0.15


acids, phenolics, and lipids may be necessaryin order to obtain an extract that is amenableto standard fractionation procedures.

Fractionation procedures can somewhat ar-bitrarily be divided into three steps: initial frac-tionation, secondary fractionation, and polish-ing. In initial processing, which deals witha large amount of extract that is not all pro-tein, materials may become soiled and may beunable to be used many times. Consequently,methods that do not require expensive reagentsor adsorbents are preferred. Classic salt frac-tionation and the less-used organic solventfractionation can achieve, if not a high degreeof purification, a useful level of concentrationand removal of much unwanted nonproteina-ceous material. Alternatively, a highly selec-tive affinity procedure may be used as the firststep, but only if the affinity material is inex-pensive to make and/or the extract is a simple,clear solution as opposed to a turbid whole-cellhomogenate.

Secondary processing achieves the mainpurification, and in difficult situations mayinvolve two or more steps. Ion-exchange(UNIT 10.10) and hydrophobic-interaction chro-matography, gel filtration (UNIT 10.9), and affin-ity techniques (UNITS 10.11B & 20.2) are themain procedures. Finally, it may be necessaryto remove traces of contaminants by “polish-ing” using high-resolution procedures such asRP-HPLC (UNITS 10.13 & 10.14) and isoelec-tric focusing (IEF; UNITS 10.3 & 10.4). Becauseevery protein has unique characteristics, it isimpossible to make general statements aboutprocedures to be followed.

Membrane-Associated and InsolubleNonrecombinant Proteins

Proteins that are not physiologically sol-uble can be purified after extracting and re-moving soluble proteins, thereby achieving asubstantial degree of purification at the ex-traction step (Fig. 10.0.4; also see Wingfield,2002, 2005). To carry out a purification itis nearly always necessary to obtain the de-sired protein in a soluble form, which will of-ten require the addition of solubilizing agentssuch as detergents. Some proteins remain in-soluble even with detergent treatment, and socan be substantially purified by removing thesoluble fractions. Some membrane-associatedproteins become partly solubilized duringbreaking up of the tissue, and recovery in theparticulate fraction may be poor. In such casesit may be best to solubilize the whole tissueby including detergent in the homogenizingbuffer. Extraction of insoluble residues using

detergents can be done differentially; someproteins are released at low detergent concen-tration, whereas others require complete solu-bilization of the membrane fraction. Suitabledetergents include nonionic (e.g., Triton) andweakly acidic types (e.g., cholic acid deriva-tives). Strongly acidic detergents such as sul-fate esters (e.g., sodium dodecyl sulfate) usu-ally cause denaturation.

Detergents can be removed either by ad-sorption of the protein on a column and sub-sequent elution without detergent, by use ofspecial detergent-adsorbing beads, or even byextraction with nonmiscible organic solventsin which the detergent partitions. On the otherhand, many membrane proteins require thepresence of detergent at all times in order toremain in solution and in a native conforma-tion. These include most integral membraneproteins, for example cytochrome P450, trans-membrane receptors, and transporters. Themost sensitive proteins require a particularcombination of natural lipids (in addition to thedetergent) to maintain structural integrity. Pu-rification methods include most of those usedfor soluble proteins, but some techniques arenot recommended if detergent is needed at alltimes. For instance, ammonium sulfate pre-cipitation will often cause a detergent-proteincomplex to come out of solution and floatrather than sink on centrifugation; this can beuseful, but the “floatate,” when redissolved,may have a high detergent content. Hydropho-bic chromatography can be very useful, asmembrane proteins are naturally hydrophobic.

Integral membrane proteins that are com-pletely insoluble in normal detergents may besolubilized by denaturation using compoundssuch as sodium dodecyl sulfate and guani-dine hydrochloride. Some cross-linked pro-teins such as elastin are not soluble withoutdisruption of the covalent linkages.

WHAT THIS CHAPTER COVERSThis chapter consists of protocols that per-

mit researchers to obtain answers to the ques-tions described above: How much protein isthere? Is the protein pure? Does the proteinhave subunits? How many protein subunits arethere? How is the protein isolated? How canthe protein be synthesized in vitro? How can aprotein be biosynthetically labeled? How canamino-terminal sequences of scarce proteinsbe determined? How can internal sequences bederived from N-terminally blocked proteins?

As indicated in Figure 10.0.5, the answer tothe first question is provided by colorimetricand spectrophotometric methods (UNIT 10.1A)

Introduction

10.0.16


Figure 10.0.4 Purification scheme for membrane-associated and poorly soluble proteins (nonre-combinant). An initial purification can be achieved by isolation of organelles containing the desiredprotein. Membrane proteins are normally solubilized with a nonionic detergent, although looselyassociated proteins may be extracted without detergent at high pH, with EDTA, or with smallamounts of an organic solvent such as n-butanol. Normal fractionation procedures may needsome modification if the detergent is required throughout to maintain the integrity of the protein.

and by amino acid analysis (UNIT 10.1B). Theamount of protein can also be determined bycomparing the staining intensity of an un-known protein to the staining intensity ofprotein standards separated by either one-or two-dimensional polyacrylamide gel elec-trophoresis (PAGE; UNITS 10.2A-10.4). UNIT 10.5describes the process required for digital imag-ing of one- and two-dimensional gels fromimage capture using scanners to image ma-nipulation and analysis using advanced imag-

ing software. Digital imaging is an immenselyuseful technique for analyzing changes in pro-tein expression and is a mainstay for func-tional genomic analysis. Proteins can alsobe stained while they are still within thepolyacrylamide gel (UNIT 10.6) or after theyhave been transferred to a blot transfer mem-brane (UNITS 10.7 & 10.19). In addition, protein-specific monoclonal or polyclonal antibodiescan be used for detection by immunoblotting(UNIT 10.8).

Analysis ofProteins

10.0.17


Figure 10.0.5 Quantitation of proteins.

Another method for quantifying a protein(or peptide) in a sample is the radioimmunoas-say (RIA), which is presented as a solution-based assay in UNIT 10.24. A solution RIA de-termines the amount of a specific protein inan unknown sample by comparison to a stan-dard curve developed using known amounts ofthe same protein. The amount of protein in theunknown sample and standards is determinedbased on the protein’s ability to compete withradiolabeled protein (the tracer) for binding toan antibody against the protein. A secondaryantibody and PEG are used to precipitate theantibody-protein complexes. Because this is acompetition assay, the amount of radioactiv-ity in the complex is inversely proportional tothe amount of protein in the sample. Underideal conditions, an RIA is capable of measur-ing picograms of antigen, but the tracer mustbe radiolabeled at a high specific activity. Ra-dioiodine (either 125I or 131I) is the label ofchoice. The solid-phase RIA (UNIT 11.17) mayalso be used for proteins and polypeptides, butis not appropriate for peptides, since the solid-phase method requires that two antigenic sitesbe present in the antigen being measured. Al-

though two or more antigenic sites are likelyin the case of proteins and polypeptides, theyare unlikely in the case of short peptides.

The answers to the next three questionscome from an analysis of the data derivedfrom a combination of electrophoresis andchromatography—i.e., conventional gel filtra-tion or size-exclusion high-performance liquidchromatography (HPLC; see Fig. 10.0.6). Fora protein without subunits or a protein withidentical subunits, detection of a single proteinband after one-dimensional gel electrophore-sis under denaturing conditions or a singlespot after two-dimensional gel electrophore-sis indicates that the protein is pure. If theprotein consists of multiple subunits of dif-ferent molecular sizes, purity is confirmed bydetecting a single stainable band after gel elec-trophoresis under nondenaturing conditions.Once the protein is demonstrated to be pure,an estimate of the molecular size of the proteinis made by comparing the elution volume ofthe protein from a conventional gel-filtrationor high-performance size-exclusion columnto the elution volumes of standard proteins.An estimate of the size of the subunits can

Introduction

10.0.18


Figure 10.0.6 Analysis of protein purity, molecular weight, and subunit structure.

be determined by subsequent electrophoresisunder denaturing conditions. The number ofeach subunit is then deduced by comparingthe molecular size of “native” (i.e., nondena-tured) protein and the molecular size(s) of thesubunit(s).

To determine how a protein or protein frag-ment should be isolated, the following fac-tors must be considered: (1) the amount ofa protein in the available starting material,(2) the cost of preparing starting material(e.g., cell culture, fermentation, or organs)and the cost of labor, (3) the molecular sizeof the protein, and (4) the physical prop-erties of the protein. In most cases a pro-tein is being isolated and purified in orderto ascertain partial protein sequence informa-tion by automated Edman degradation usinga commercially available protein sequencer.However, almost all proteins are isolated bya combination of conventional chromatogra-phy, HPLC, and electrophoresis (Fig. 10.0.7).

Each of these isolation methods is discussedin detail in the following sections of thisintroduction.

Both one- and two-dimensional gel elec-trophoresis are high-resolution separationmethods, yielding protein whose sequencecan be determined after electroblotting ontopolyvinylidene difluoride (PVDF) membranefilters (which are compatible with a gas-phaseprotein sequencer; UNIT 10.19) or capillaryelectrophoresis (UNITS 10.14 & 10.20). In mostcases, electrophoretic methods are used afterseveral successive modes of conventionalchromatography or HPLC have been used topurify progressively a given protein from acrude protein mixture. However, if the pro-tein is separated under denaturing conditions,the biological activity of a desired protein willlikely be lost. This is a reason for utilizinggel electrophoresis last when purifying a pro-tein whose identity is based on a functionalassay.

Analysis ofProteins

10.0.19


Figure 10.0.7 Isolation of proteins and polypeptide fragments.

Conventional chromatography includes gelfiltration (UNIT 10.9), ion exchange (UNIT 10.10),immunoaffinity (UNIT 10.11A), affinity on im-mobilized dyes (UNIT 10.11B), affinity on im-mobilized ligands (UNITS 10.15, 16.6–16.8 & 24.5),and hydrophobic interaction. All of thesemethods can accommodate large amounts ofcrude starting material. Because gel-filtrationchromatography has a greater separating rangethan comparable size-exclusion HPLC, gel fil-tration is preferred for separating proteins ofsimilar molecular sizes. None of these chro-matographic modes will cause protein denat-uration, although contaminating proteases incrude protein mixtures can always lead todegradation of a protein during purification.

HPLC is an essential tool for the purifica-tion of proteins. Start-up procedures, samplepreparation, preparation of mobile phase, set-up of the HPLC instrument, pump priming,gradient design, and sample injection are cov-ered in detail in UNIT 10.12. Eight basic modesof HPLC are currently used for protein pu-rification: size exclusion (UNIT 10.13), ion ex-change (UNIT 10.13), normal phase (UNIT 10.13),reversed phase (UNITS 10.13 & 10.14), hydropho-bic interaction (UNIT 10.13), hydrophilic inter-action (UNIT 10.13), immobilized metal affin-ity (UNIT 10.13), and biospecific/biomimeticaffinity (UNIT 10.13). All these HPLC meth-ods are equivalent to their counterparts inconventional chromatography. However, the

Introduction

10.0.20


advantages of HPLC over conventional chro-matography are that small amounts (

Analysis ofProteins

10.0.21


understood for most proteins, and over 400distinct chemical modifications have been de-scribed. The detection and chemical identifi-cation of such modifications is one of the fron-tiers of cell biology.

UNIT 10.23 describes differential gel elec-trophoresis (DIGE), a technique for labelingproteins with three unique cyanine fluorescentdyes (called Cy2, Cy3, and Cy5), which differin their fluorescence spectral properties. TheCy dyes are covalently attached to the proteinsin a mixture using an active ester couplingprocedure (using N-hydroxysuccinimide esterderivatives of the various Cy dyes), which re-sults in minimal (1% to 2%) labeling of the ε-amino group of lysine residues of the variousproteins in a mixture. Because the net chargeof a lysine residue or a Cy dye molecule is −1,there is no appreciable change in the isoelec-tric point. In addition, there is no detectablechange in the molecular mass of the labeledproteins, unless they are very low molecu-lar mass, since the mass of a Cy molecule is∼500. The labeled proteins from different pro-tein samples are analyzed by 2-D electrophore-sis using a multiplex analysis format (i.e., twoor three samples are mixed and applied to thesame gel) and the individual separation pat-terns (between two or among three Cy-labeledmixtures) are revealed by scanning the gelswith excitation wavelengths corresponding tothe maxima of the different dyes. Becausethe Cy dyes are detected with the sensitiv-ity of silver-stained gels, the DIGE methodis also useful for comparing the expression ofrecombinant proteins under different expres-sion conditions, protein mixtures fractionatedby conventional chromatography or HPLC,and membrane protein mixtures from differentstrains. Importantly, Cy dye-labeled proteinseluted from gels or separated chromatograph-ically are compatible with subsequent massspectrometric analysis.

UNIT 10.24 describes the use of solutionradioimmunoassays to determine picomolaramounts of proteins in complex biologicalsamples. The method requires the initial pu-rification of a sufficient quantity of the un-known protein so that it may be used as animmunogen for generating polyclonal or mon-oclonal anti-protein antibodies, as radioiod-inated protein tracer, and as control proteinfor constructing a standard curve. Proteinsare routinely radioiodinated in trace amountsby either Bolton-Hunter reagent (labelinglysyl residues) or by lactoperoxidase (labelingtyrosine residues).

CONCLUSIONThrough proteomics, structural genomics,

and cell biology, the post-genomic era is ad-dressing aspects of the protein problem thatare now tractable. The currently intractable as-pects will only be elucidated when new tech-nologies are developed that move us beyondthe limitations of purification and analysismethods that are the state of the art describedin this chapter. Without a concerted effort tomove beyond the limitations of contemporarymethods, which were developed to isolate in-dustrial quantities of proteins (beginning inthe 1940s) and were premised upon proteinsexisting as solitary molecules with invariantfunction (the “one gene, one protein” hypoth-esis, which is largely the realm of molecularbiology), a real understanding of the diver-sity, distribution, and functions of proteins willremain an illusory goal.

LITERATURE CITEDColigan, J.E., Dunn, B.M., Speicher, D.W., and

Wingfield, P.T. (eds.). 2006. Current Protocols inProtein Science. John Wiley & Sons, Hoboken,N.J..

Oka, M., Yang, Y.S., Nagata, S., Esaki, N., Tanaka,M., and Soda, K. 1989. Overproduction ofthermostable leucine dehydrogenase of Bacil-lus stearothermophilus and its one-step purifi-cation from recombinant cells of Escherichiacoli. Biotechnol. Appl. Biochem. 11:307-316.

Pain, R.H. 1995. Overview of protein folding.In Current Protocols in Protein Science (J.E.Coligan, B.M. Dunn, H.L. Ploegh, D.W.Speicher, and P.T. Wingfield, eds.) pp. 6.4.1-6.4.7. John Wiley & Sons, New York.

Palmer, I. and Wingfield, P.T. 2004. Preparation andextraction of insoluble (inclusion-body) proteinsfrom Escherichia coli. In Current Protocols inProtein Science (J.E. Coligan, B.M. Dunn, D.W.Speicher, and P.T. Wingfield, eds.) pp. 6.3.1-6.3.18. John Wiley & Sons, Hoboken, N.J.

Sassenfeld, H.M. 1990. Engineering proteins forpurification. Trends Biotechnol. 8:88-93.

Walter, H. and Johansson, G. (eds.) 1994. Aqueoustwo-phase systems. Methods Enzymol. 228:1-725.

Wingfield, P.T. 1995. Use of protein foldingreagents. In Current Protocols in Protein Science(J.E. Coligan, B.M. Dunn, D.W. Speicher, andP.T. Wingfield, eds.) pp. A.3A.1-A.3A.4. JohnWiley & Sons, New York.

Wingfield, P.T. 2002. Overview of the purifi-cation of recombinant proteins produced inEscherichia coli. In Current Protocols inProtein Science (J.E. Coligan, B.M. Dunn, H.L.Ploegh, D.W. Speicher, and P.T. Wingfield,eds.) pp. 6.1.1-6.1.37. John Wiley & Sons,New York.

Introduction

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Wingfield, P.T. 2005. Preparation of solubleproteins from Escherichia coli. In CurrentProtocols in Protein Science (J.E. Coligan, B.M.Dunn, D.W. Speicher, and P.T. Wingfield, eds.)pp. 6.2.1-6.2.22. John Wiley & Sons, Hoboken,N.J.

Wingfield, P.T., Palmer, I., and Liang, S.-M. 1995.Folding and purification of insoluble (inclusion-body) proteins from Escherichia coli. InCurrent Protocols in Protein Science (J.E.Coligan, B.M. Dunn, H.L. Ploegh, D.W. Spe-icher, and P.T. Wingfield, eds.) pp. 6.5.1-6.5.27.John Wiley & Sons, New York.

Zeilstra-Ryalls, J., Fayet, O., and Georgopoulos, C.1991. The universally-conserved GroE (Hsp60)chaperonins. Annu. Rev. Microbiol. 45:301-325.

KEY REFERENCESColigan et al., 2006. See above.Companion to this manual giving detailed proto-cols for many procedures commonly used in proteinchemistry, including general handling, purification,and analysis.

Deutscher, M.P. (ed.) 1990. Guide to proteinpurification. Methods Enzymol. 182:1-894.

Extensive collection of purification methods, withsome general protocols and examples.

Janson, J.-C. and Ryden, L.G. 1989. Protein Pu-rification: Principles, High Resolution Methods,and Applications. VCH Publishers, New York.

A useful collection of methods and examples.

Kennedy, J.F. and Cabral, J.M. (eds.) 1993. Re-covery Processes for Biological Materials. JohnWiley & Sons, New York.

A useful introduction to the problems of large-scalemethods.

Kenny, A. and Fowell, S. (eds.) 1992. Practical pro-tein chromatography. Methods Mol. Biol. 11:1-327.

Extensive descriptions of affinity chromatographictechniques with protocols and recipes.

Scopes, R.K. 1993. Protein Purification, Principlesand Practice, 3rd ed. Springer-Verlag, New Yorkand Heidelberg.

General principles of all the main techniques usedin purifying proteins. A useful laboratory hand-book; does not include recipes or procedures forspecific proteins.

Contributed by R.K. ScopesLa Trobe UniversityBundoora, Australia

John A. SmithUniversity of AlabamaBirmingham, Alabama

SECTION IQUANTITATION OF PROTEINSThe success or failure of protein-centered projects can frequently be traced to the qualityof the analytical procedures used to characterize the sample at different stages. Qualitativeand quantitative analysis can aid in definition of the sample for the purpose of designingseparations. A knowledge of the properties of the desired protein (e.g., whether it has ahigh aromatic amino acid content) can suggest methods of analysis that will help locatethe desired protein in a complex mixture. Establishing the properties of an isolated proteincreates benchmarks that future researchers can use to evaluate their protocols and finalproduct. Accurate quantitation of the amount of protein at the beginning, middle, andend of a series of steps is the only valid way to evaluate the overall yield of a procedure.The observation of significant loss of protein, without a substantial increase in the purityof the desired protein, following a particular purification procedure would indicate thatthe procedure should be omitted or revised.

Several spectroscopic procedures for characterizing protein samples are described inUNIT 10.1A. Measuring the absorbance of the aromatic amino acids in a protein at differencewavelengths yields a very useful measure of protein concentration. This is non-destructiveand requires very little sample or time. A more qualitative, but much more sensitive,evaluation is provided by fluorescence spectroscopy. Quantitation of the amount ofprotein contained in a solution also can be conveniently accomplished using colorimetricmethods. The Bradford and the Lowry methods are the most frequently used and reliableprocedures. The method of choice is the Bradford method, which is easy and rapid tocomplete.

Another approach is amino acid analysis—qualitative analysis to determine purity andquantitative analysis to provide concentrations—both of which are presented in UNIT 10.1B.Procedures are also given for calculating amino acid composition from primary analyticaldata. Significant advances that have improved the precision and sensitivity of amino acidanalysis have reinvigorated this method, which had for some years been neglected.

Current Protocols in Molecular Biology (2006)Copyright C© 2006 by John Wiley & Sons, Inc.

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Supplement 76

UNIT 10.1ASpectrophotometric and ColorimetricDetermination of Protein Concentration

This unit describes spectrophotometric and colorimetric methods for measuring theconcentration of a sample protein in solution. In Basic Protocol 1, absorbance measuredat 280 nm (A280) is used to calculate protein concentration by comparison with a standardcurve or published absorptivity values for that protein (a280). In Alternate Protocol 1,absorbance measured at 205 nm (A205) is used to calculate the protein concentration. TheA280 and A205 methods can be used to quantitate total protein in crude lysates and purifiedor partially purified protein. Both of these methods are simple and can be completedquickly. The A280 method is the most commonly used. The A205 method can detect lowerconcentrations of protein and is useful for dilute protein samples, but is more susceptibleto interference by reagents in the protein sample than the A280 method. Basic Protocol 2uses a spectrofluorometer or a filter fluorometer to measure the intrinsic fluorescenceemission of a sample solution; this value is compared with the emissions from standardsolutions to determine the sample concentration. The fluorescence emission method isused to quantitate purified protein. This simple method is useful for dilute protein samplesand can be completed in a short amount of time. The Bradford colorimetric method, basedupon binding of the dye Coomassie brilliant blue to an unknown protein, is presentedin Basic Protocol 3; the Lowry method, which measures colorimetric reaction of tyrosylresidues in an unknown, is given in Alternate Protocol 2.

BASICPROTOCOL 1

USING A280 TO DETERMINE PROTEIN CONCENTRATION

Determination of the concentration of protein in a solution by absorbance measurementsat 280 nm (A280) is based on the inherent absorbance of UV light by the aromaticamino acids tryptophan and tyrosine, as well as by cystine (disulfide-bonded cysteineresidues). The measured absorbance of a protein sample solution is used to calculate theconcentration either from its published absorptivity at 280 nm (a280) or by comparisonwith a calibration curve prepared from measurements with standard protein solutions.This assay can be used to quantitate solutions with protein concentrations of 20 to3000 µg/ml.

Materials

3 mg/ml spectrophotometric standard protein solution (see recipe; optional)Sample proteinSpectrophotometer with UV lampQuartz cuvette

1. For calibrating with standards, use the 3 mg/ml standard protein solution to preparedilutions of 20, 50, 100, 250, 500, 1000, 2000, and 3000 µg/ml in the same solventused to prepare the sample protein. Prepare a blank consisting of solvent alone.

Ideally, for purified or partially purified protein, the protein standard should have anaromatic amino acid content similar to that of the sample protein. For the total proteinof a crude lysate, an average absorptivity (step 5a) can be used to calculate the proteinconcentration directly from the measured A280 without standards. Bovine serum albumin(BSA) is commonly used as a standard for spectrophotometric quantitation of proteinconcentration of crude lysates. However, the a280 for BSA is 0.6 ml/mg, which is lessthan one half the calculated average a280 (1.3 ml/mg) from 80 proteins (Pace et al.,1995). Therefore, use of BSA as a standard for protein quantitation could result in anunderestimation of protein concentration of a given sample that has a greater aromaticamino acid content than BSA.

Contributed by Michael H. Simonian and John A. SmithCurrent Protocols in Molecular Biology (2006) 10.1A.1-10.1A.9Copyright C© 2006 by John Wiley & Sons, Inc.

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Supplement 76

Spectrophoto-metric and

ColorimetricDetermination of

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10.1A.2


2. Turn on the UV lamp of the spectrophotometer and set the wavelength to 280 nm.Allow the instrument to warm up 30 min before taking measurements.

3. Zero the spectrophotometer with the solvent blank.

When removing solutions from the cuvette, do not remove the cuvette from the spec-trophotometer. Leaving the cuvette in the spectrophotometer between readings will resultin greater reproducibility of the absorbance readings. Use of an aspirator pump and thintubing is the ideal method for completely removing the solutions from the cuvette. If thisis not available, use a pipettor and tip to remove the solutions from the cuvette.

4. Measure the absorbance of the protein standard and unknown solutions.

If the A280 of the sample protein is >1.5, dilute the sample further in the same solvent andmeasure the A280 again.

5a. If the a280 of the protein is known: Calculate the unknown sample concentration fromits absorbance value using the following equation, where a280 has units of ml/mg cmand b is the path length in cm.

Because most cuvettes have a 1-cm path length, the b term can usually be ignored inthis equation. For calculation of the protein content of crude lysates, an average a280 of1.3 ml/mg cm can be used. This value was determined from absorptivities of 80 proteins(Pace et al., 1995).

5b. If standard solutions are used for quantitation: Create a calibration curve by eitherplotting or performing regression analysis of the A280 versus concentration of thestandards. Use the absorbance of the sample protein to determine the concentrationfrom the calibration curve.

ALTERNATEPROTOCOL 1

USING A205 TO DETERMINE PROTEIN CONCENTRATION

Determination of protein concentration by measurement of absorbance at 205 nm (A205)is based on absorbance by the peptide bond. The concentration of a protein sample isdetermined from the measured absorbance and the absorptivity at 205 nm (a205). Thisassay can be used to quantitate protein solutions with concentrations of 1 to 100 µg/mlprotein.

Additional Materials (also see Basic Protocol 1)

Brij 35 solution: 0.01% (v/v) Brij 35 (Sigma) in an aqueous solution appropriatefor dissolving or diluting the sample protein

1. Dissolve or dilute the protein sample in Brij 35 solution.

2. Turn on the UV lamp of the spectrophotometer and set the wavelength to 205 nm.Allow the instrument to warm up 30 min before taking measurements.

3. Zero the spectrophotometer with the Brij 35 solution alone.

4. Measure the absorbance of the sample protein.

When removing solutions from the cuvette, do not remove the cuvette from the spec-trophotometer. Leaving the cuvette in the spectrophotometer between readings will resultin greater reproducibility of the absorbance readings. Use of an aspirator pump and thintubing is the ideal method for completely removing the solutions from the cuvette. If thisis not available, use a pipettor and tip to remove the solutions from the cuvette.

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5a. If the a205 of the protein is known: Use the equation relating A280 and proteinconcentration (see Basic Protocol 1, step 5a) to calculate the concentration of thesample protein, except substitute the appropriate values for A205 and a205.

5b. If the a205 is not known: Estimate the concentration of the sample protein from itsmeasured absorbance using the following equation, where the absorptivity value, 31,has units of ml/mg cm and b is the path length in cm.

The absorptivity value of 31 ml/mg cm is an average derived from measurement of tenpurified proteins (Scopes, 1974). The proteins were first dried and then several dilutionswere made in buffer; the a205 for each protein was calculated from the absorbancereadings of the dilutions.

BASICPROTOCOL 2

USING FLUORESCENCE EMISSION TO DETERMINE PROTEINCONCENTRATION

Protein concentration can also be determined by measuring the intrinsic fluorescencebased on fluorescence emission by the aromatic amino acids tryptophan, tyrosine, and/orphenylalanine. Usually tryptophan fluorescence is measured. The fluorescence intensityof the protein sample solution is measured, and the concentration of the protein samplesolution calculated from a calibration curve based on the fluorescence emission of stan-dard solutions prepared from the purified protein. This assay can be used to quantitateprotein solutions with concentrations of 5 to 50 µg/ml.

Materials

Spectrophotometric protein standard solution (see recipe) prepared using thepurified protein

Sample proteinSpectrofluorometer or filter fluorometer with an excitation cutoff filter at ≤285 nm

and an emission filter at >320 nmQuartz fluorometer cuvette

1. Prepare dilutions of the purified protein at 5, 7.5, 10, 25, and 50 µg/ml in the samesolvent as the sample protein. Prepare a blank consisting of solvent alone.

2. Turn on the lamp of the instrument and allow it to warm up 30 min before takingmeasurements.

If a spectrofluorometer is used, set the excitation wavelength to 280 nm and the emissionwavelength to between 320 and 350 nm. If the exact emission wavelength is not known,determine it empirically by scanning the standard solution with the excitation wavelengthset to 280 nm. If the instrument is a filter fluorometer, use an excitation cutoff filter at≤285 nm and an emission filter at >320 nm.

3. Zero the instrument with the solvent blank.

4. Measure the fluorescence of the protein standard and sample protein solutions.

5. Create a calibration curve by either plotting or performing regression analysis of thefluorescence intensity versus concentration of the standards. Using the fluorescenceintensity of the sample protein, determine the concentration from the calibrationcurve.

Fluorescence emission is a linear function of concentration only over a limited range.

Spectrophoto-metric and

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10.1A.4


BASICPROTOCOL 3

USING THE BRADFORD METHOD TO DETERMINE PROTEINCONCENTRATION

The Bradford method depends on quantitating the binding of a dye, Coomassie brilliantblue, to an unknown protein and comparing this binding to that of different amounts ofa standard protein, usually BSA. It is designed to quantify 1 to 10 µg protein. Proteindeterminations in the range of 10 to 100 µg may be carried out by increasing the volumeof the dye solution 5-fold and using larger tubes.

Materials

Colorimetric standard protein solution (0.5 mg/ml BSA; see recipe)0.15 M NaClCoomassie brilliant blue solution (see recipe)1 ml, 1-cm-path-length microcuvette

1. Into eight microcentrifuge tubes place duplicate aliquots of 0.5 mg/ml BSA (5,10, 15, and 20 µl) and dilute each to 100 µl with 0.15 M NaCl. Into two moremicrocentrifuge tubes, place 100 µl each of 0.15 M NaCl; these are blank tubes.

2. Add 1 ml Coomassie brilliant blue solution and vortex. Allow to stand 2 min at roomtemperature.

3. Determine the A595 using a 1-cm-path-length (1 ml) microcuvette and make a stan-dard curve by plotting absorbance at 595 nm versus protein concentration. Determinethe absorbance for the unknown and use the standard curve to determine the concen-tration of protein in the unknown.

If the unknown protein concentration is too high, dilute the protein, assay a smalleraliquot of the unknown, or generate another standard curve in a higher concentrationrange (e.g., 10 to 100 µg).

ALTERNATEPROTOCOL 2

USING THE LOWRY METHOD TO DETERMINE PROTEINCONCENTRATION

The Lowry method depends on quantitating the color obtained from the reaction of Folin-Ciocalteu phenol reagent with the tyrosyl residues of an unknown protein and comparingthis color value to the color values derived from a standard curve of a standard protein,usually BSA. This assay is desi

Documents

CHAPTER 10 Analysis of Proteinssun-lab.med.nyu.edu/.../CPMB.Ch.10.Analysis.Proteins.pdf(bioinformatics and sequence databases), Chapter 20 (protein-protein interactions), Chapter21(chromatinassemblyandanalysis),