Preface The explosion of work and interest in molecular biology in recent years has made protein purification something of a lost art, especially among younger biochemists and molecular biologists. At the same time, many of the more interesting biological problems have now reached a stage that requires work with purified proteins and enzymes. This has led to a situation in which many important studies stop at the demonstration of a physiological effect, and are not carried through to an understanding of the proteins responsible for the phenomenon. For these reasons a methods manual dealing with all aspects of protein purification should be a valuable addition to the Methods in Enzymology series and should be extremely useful to the scientific community. Although techniques for protein purification have been included in a few volumes in this series in the past, this Guide brings together in one source up-to-date procedures for purifying, characterizing, and working with proteins and enzymes. The volume begins with introductory chap- ters describing the rationale for studying proteins and enzymes with strat- egies for their purification, is followed by contributions that familiarize the reader with procedures for working with proteins and enzymes, and proceeds to describe in detail methods for their purification and character- ization. Useful immunological procedures and other techniques that aid in the study of proteins are also included. In addition to the methods articles that make up the bulk of the Guide, a few retrospective chapters by eminent biochemists, which describe one of their famous studies in order to give a feeling for the "art" of enzyme purification that goes beyond techniques and mechanical procedures, have been included. The Guide is a self-contained volume covering all the important proce- dures for purifying proteins, as well as other more specialized techniques. However, to stay within the confines of a single volume, some details are dealt with by reference to other works, but these have been kept to a minimum. It is hoped that this volume will satisfy the needs of both the novice in protein purification and the more experienced researcher. MURRAY P. DEUTSCHER xiii Contributors to Volume 182 Article numbers are in parentheses following the names of contributors. Affiliations listed are current. PATRICK ARGOS (56), European Molecular Biology Laboratory, 6900 Heidelberg, Federal Republic of Germany JOHN S. BLANCHARD (4), Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 MARGARET K. BRADLEY (10), Department of Pathology, Dana-Farber Cancer Insti- tute and the Harvard Medical School, Boston, Massachusetts 02115 ROMAN M. CHICZ (32), Department of Bio- chemistry and Molecular Biology, Har- vard University, Cambridge, Massachu- setts 02138 CHRIS CIVALIER (39), Department of Micro- biology, University of Connecticut Health Center, Farmington, Connecticut 06032 MILLARD CULL (12), Department of Bio- chemistry, Biophysics and Genetics, Uni- versity of Colorado Health Sciences Cen- ter, Denver, Colorado 80262 AsIs DAS (9), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 MURRAY P. DEUTSCHER (3, 8, 57), Depart- ment of Biochemistry, University of Con- necticut Health Center, Farmington, Connecticut 06032 JOHN DAVID DIGNAM (15), Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699 BONNIE S. DUNBAR (34, 49 51), Depart- ment of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 SHLOMO EISENBERG (39), Department of Microbiology, University of Connecticut Health Center, Farmington, Connecticut 06032 SASHA ENGLARD (22, 47), Department of ix Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461 GARY L. FIRESTONE (52), Department of Molecular and Cell Biology and Cancer Research Laboratory, University of Cali- fornia at Berkeley, Berkeley, California 94720 STEPHEN C. FRANCESCONI (39), Depart- ment of Microbiology, University of Con- necticut Health Center, Farmington, Connecticut 06032 DAVID E. GARFIN (33, 35), Chemical Divi- sion, Research Products Group, Bio-Rad Laboratories, Incorporated, Richmond, California 94804 PETER GEGENHEIMER (14), Departments of Botany and Biochemistry, University of Kansas, Lawrence, Kansas 66045 CRAIG GERARD (40), Department of Pediat- rics, Harvard Medical School, Children's Hospital Medical Center, Boston, Massa- chusetts 02115 LALLAN GIRl (31), Quality Control Depart- ment, Connaught Laboratories, Inc., Swiftwater, PA 18370 MARINA J. GORBUNOFF (26), Graduate De- partment of Biochemistry, Brandeis Uni- versity, Waltham, Massachusetts 02254 MICHAEL G. HARRINGTON (37), Biology De- partment, California Institute of Technol- ogy, Pasadena, California 91125 DONNA L. HARTLEY (20), Centre Interna- tional de Recherche Daniel Carasso, 92350 Le Plessis-Robinson, Paris, France LEONARD M. HJELMELAND (19, 21), De- partments of Ophthalmology and Biologi- cal Chemistry, School of Medicine, Uni- versity of California, Davis, Davis, California 95616 X CONTRIBUTORS TO VOLUME 182 I. BARRY HOLLAND (11), Department of Ge- netics, University of Leicester, Leicester LEI 7RH, England B. L. HORECKER (59), Department of Bio- chemistry, Cornell University Medical College, New York, New York 10021 KENNETH C. INGHAM (23), Biochemistry Laboratory, American Red Cross Hol- land Laboratories, Rockville, Maryland 20855 S. MICHAL JAZWlNSKI (13), Department of Biochemistry and Molecular Biology, Louisiana State University Medical Cen- ter, New Orleans, Louisiana 70112 RALPH C. JUDD (46), Division of Biological Sciences, University of Montana, Mis- soula, Montana 59812 ROBERT M. KENNEDY (27), Membrex Incor- porated, Garfield, New Jersey 07026 BRENDAN KENNY (1 l), Department of Ge- netics, University of Leicester, Leicester LEI 7RH, England HITOMI KIMURA (34), Department of Bio- chemistry, State University of New York at Stony Brook, Stony Brook, New York 11794 ARTHUR KORNBERG (1, 58), Department of Biochemistry, Stanford University, Stan- ford, California 94305 THOMAS M. LAUE (42, 43), Department of Biochemistry, University of New Hamp- shire, Durham, New Hampshire 03824 STUART LINN (2), Division of Biochemistry and Molecalar Biology, University of Cal- ifornia, Berkeley, Berkeley, California 94720 EDWARD A. MADDEN (16), Department of Biology, University of Indianapolis, Indi- anapolis, Indiana 46227 FIONA A. O. MARSTON (20), Celltech Lim- ited, Slough, Berkshire SL1 4EN, En- gland CHRISTOPHER K. MATHEWS (41), Depart- ment of Biochemistry and Biophysics, Or- egon State University, Corvallis, Oregon 97331 PAUL MATSUDAIRA (45), Whitehead lnsti- tute, Massachusetts Institute of Technol- ogy, Cambridge, Massachusetts 02142 CHARLES S. MCHENRY (12), Department of Biochemistry, Biophysics and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262 MARK G. MCNAMEE (38), Department of Biochemistry and Biophysics, University of California, Davis, California 95616 CARL R. MERRIL (36), Laboratory of Bio- chemical Genetics, National Institute of Mental Health, Bethesda, Maryland 20892 KIVIE MOLDAVE (61), Department of Biol- ogy, University of California, Santa Cruz, Santa Cruz, California 95064 JUDITH M. NEUGEBAUER (18), Department of Chemistry and Institute of Colloid and Surface Science, Clarkson University, Potsdam, New York 13676 DAVID OLLIS (48), Department of Biochem- istry, Molecular Biology and Cell Biology, Northwestern University, Evanston, Illi- nois 60208 STEVEN OSTROVE (29, 30), Davy McKee Corporation, Berkeley Heights, New Jer- sey 07922 JuRlS OZOLS (17, 44), Department of Bio- chemistry, University of Connecticut Health Center, Farmington, Connecticut 06032 CHARLES W. PARKER (53, 54), Department of Medicine and Microbiology, Washing- ton University School of Medicine, St. Louis, Missouri 63110 ANDREAS PLOCKTHUN (11), Gen-Zentrum der Universitat Miinchen, Max-Planck-In- stitut flir Biochemie, D-8033 Martinsried, Munich, Federal Republic of Germany THOMAS POHL (7), Abteilungfiir Molekulare Neuroendokrinologie, Max-Planck-lnsti- tut fiir Experimentelle Medizin, 3400 G6t- tingen, Federal Republic of Germany FRED E. REGNIER (32), Department of Bio- chemistry, Purdue University, West La- fayette, Indiana 47907 CONTRIBUTORS TO VOLUME 182 xi DAVID G. RHODES (42, 43), Biomolecular Structure Analysis Center, Department of Radiology, University of Connecticut Health Center, Farmington, Connecticut O6O32 EDWARD F. ROSSOMANDO (5, 24), Depart- ment of BioStructure and Function, Uni- versity of Connecticut Health Center, Farmington, Connecticut 06032 ERIc D. SCHWOEBEL (49), Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 SAM SEIFTER (22, 47), Department of Bio- chemistry, Albert Einstein College of Medicine, Bronx, New York 10461 SHERI M. SKINNER (50), Department of Cell Biology, Baylor College of Medicine, Houston, Texas 77030 PAUL A. SRERE (41), Research Service, De- partment of Veteran Affairs, University of Texas Southwestern Medical Center, Dal- las, Texas 75216 EARL R. STADTMAN (60), National Heart, Lung and Blood Institute, National Insti- tutes of Health, Bethesda, Maryland 20892 BORIS STEIPE (l 1), Gen-Zentrum der Un- iversitat Miinchen, Max-Planck-Institut fiir Biochemie, D-8033 Martinsried, Mu- nich, Federal Republic of Germany EARLE STELLWAGEN (25, 28), Department of Biochemistry, University of Iowa, Iowa City, Iowa 52242 VINCENT S. STOLE (4), Department of Bio- chemistry, Albert Einstein College of Medicine, Bronx, New York 10461 BRIAN STORRIE (16), Biochemistry Depart- ment, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24060 CHRISTA M. STOSCHECK (6), Department of Medicine, Division of Dermatology, Vet- erans Administration, Nashville, Tennes- see 37212 THOMAS C. THOMAS (38), Department of Biochemistry and Biophysics, University of California, Davis, Davis, California 95616 THERESE M. TIMMONS (34, 51), Department of Cell Biology, Baylor College of Medi- cine, Houston, Texas 77030 O. TSOEAS (59), Laboratory of Biological Chemistry, University of loannina Medi- cal School, Ioannina, CaR 453 32 Greece SCOTT S. WALKER (39), Department of Mi- crobiology, University of Connecticut Health Center, Farmington, Connecticut 06032 SHELLY WEISS (30), New Brunswick Scien- tific, Edison, New Jersey 08818 STEPHEN WHITE (48), Department of Biol- ogy, Brookhaven National Laboratory, Upton, New York 11973 SANDRA D. WINGUTH (52), Department of Ophthalmology, Ocular Oncology Unit, University of California at San Francisco, San Francisco, California 94143 JOHN M. WOZNEY (55), Genetics Institute, Incorporated, Cambridge, Massachusetts 02140 [1] WHY PURIFY ENZYMES? 1 [1] Why Purify Enzymes? By ARTHUR KORNBERG "Don't waste clean thinking on dirty enzymes" is an admonition of Efraim Racket's which is at the core of enzymology and good chemical practice. It says simply that detailed studies of how an enzyme catalyzes the conversion of one substance to another is generally a waste of time until the enzyme has been purified away from the other enzymes and substances that make up a crude cell extract. The mixture of thousands of different enzymes released from a disrupted liver, yeast, or bacterial cell likely contains several that direct other rearrangements of the starting material and the product of the particular enzyme's action. Only when we have purified the enzyme to the point that no other enzymes can be detected can we feel assured that a single type of enzyme molecule directs the conversion of substance A to substance B, and does nothing more. Only then can we learn how the enzyme does its work. The rewards for the labor of purifying an enzyme were laid out in a series of inspirational papers by Otto Warburg in the 1930s. From his laboratory in Bedin-Dahlem came the discipline and many of the methods of purifying enzymes and with those the clarification of key reactions and vitamin functions in respiration and the fermentation of glucose. War- burg's contributions strengthened the classic approach to enzymology inaugurated with Eduard Btichner's accidental discovery, at the turn of this century, of cell-free conversion of sucrose to ethanol. One tracks the molecular basis of cellular function alcoholic fermentation in yeast, gly- colysis in muscle, luminescence in a fly, or the replication of DNA by first observing the phenomenon in a cell-free system. Then one isolates the responsible enzyme (or enzymes) by fractionation of the cell extract and purifies it to homogeneity. Then one hopes to learn enough about the structure of the enzyme to explain how it performs its catalytic functions, responds to regulatory signals, and is associated with other enzymes and structures in the cell. By a reverse approach, call it neoclassical, especially popular in re- cent decades, one first obtains a structure and then looks for its function. The protein is preferably small and stable, and has been amplified by cloning or is commercially available. By intensive study of the protein and homologous proteins, one hopes to get some clues to how it functions. As the popularity of the neoclassical approach has increased, so has there Copyright © 1990 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 182 All rights of reproduction in any form reserved. 2 METHODS IN ENZYMOLOGY [1] been a corresponding decrease in interest in the classical route: pursuit of a function to isolate the responsible structure. Implicit in the devotion to purifying enzymes is the faith of a dedicated biochemist of being able to reconstitute in a test tube anything a cell can do. In fact, the biochemist with the advantage of manipulating the medium: pH, ionic strength, etc., by creating high concentrations of reactants, by trapping products and so on, should have an easier time of it. Another article of faith is that everything that goes on in a cell is catalyzed by an enzyme. Chemists sometimes find this conviction difficult to swallow. On a recent occasion I was told by a mature and well-known physical chemist that what fascinated him most in my work was that DNA replica- tion was catalyzed by enzymes ! This reminded me of a seminar I gave to the Washington University chemistry department when I arrived in St. Louis in 1953. I was describing the enzymes that make and degrade orotic acid, and began to realize that my audience was rapidly slipping away. Perhaps they had been expecting to hear about an organic synthesis of erotic acid. In a last-ditch attempt to retrieve their attention, I said loudly that every chemical event in the cell depends on the action of an enzyme. At that point, the late Joseph Kennedy, the brilliant young chairman, awoke: "Do you mean to tell us that something as simple as the hydration of carbon dioxide (to form bicarbonate) needs an enzyme?" The Lord had delivered him into my hands. "Yes, Joe, cells have an enzyme, called carbonic anhydrase. It enhances the rate of that reaction more than a million fold." Enzymes are awesome machines with a suitable level of complexity. One may feel ill at ease grappling with the operations of a cell, let alone those of a multiceUular creature, or feel inadequate in probing the fine chemistry of small molecules. Becoming familiar with the personality of an enzyme performing in a major synthetic pathway can be just right. To gain this intimacy, the enzyme must first be purified to near homogeneity. For the separation of a protein species present as one-tenth or one-hun- dredth of 1% of the many thousands of other kinds in the cellular commu- nity, we need to devise and be guided by a quick and reliable assay of its catalytic activity. No enzyme is purified to the point of absolute homogeneity. Even when other proteins constitute less than 1% of the purified protein and escape detection by our best methods, there are likely to be many millions of foreign molecules in a reaction mixture. Generally, such contaminants do not matter unless they are preponderantly of one kind and are highly active on one of the components being studied. [1] WHY PURIFY ENZYMES? 3 Only after the properties of the pure enzyme are known is it profitable to examine its behavior in a crude state. "Don't waste clean thinking on dirty enzymes" is sound dogma. I cannot recall a single instance in which I begrudged the time spent on the purification of an enzyme, whether it led to the clarification of a reaction pathway, to discovering new en- zymes, to acquiring a unique analytical reagent, or led merely to greater expertise with purification procedures. So, purify, purify, purify. Purifying an enzyme is rewarding all the way, from first starting to free it from the mob of proteins in a broken cell to having it finally in splendid isolation. It matters that, upon removing the enzyme from its snug cellular niche, one cares about many inclemencies: high dilution in unfriendly solvents, contact with glass surfaces and harsh temperatures, and expo- sure to metals, oxygen, and untold other perils. Failures are often attrib- uted to the fragility of the enzyme and its ready denaturability, whereas the blame should rest on the scientist for being more easily denatured. Like a parent concerned for a child's whereabouts and safety, one cannot leave the laboratory at night without knowing how much of the enzyme has been recovered in that day's procedure and how much of the contami- nating proteins still remain. To attain the goal of a pure protein, the cardinal rule is that the ratio of enzyme activity to the total protein is increased to the limit. Units of activity and amounts of protein must be strictly accounted for in each manipulation and at every stage. In this vein, the notebook record of an enzyme purification should withstand the scrutiny of an auditor or bank examiner. Not that one should ever regard the enterprise as a business or banking operation. Rather, it often may seem like the ascent of an un- charted mountain: the logistics like those of supplying successively higher base camps. Protein fatalities and confusing contaminants may resemble the adventure of unexpected storms and hardships. Gratifying views along the way feed the anticipation of what will be seen from the top. The ultimate reward of a pure enzyme is tantamount to the unobstructed and commanding view from the summit. Beyond the grand vista and thrill of being there first, there is no need for descent, but rather the prospect of even more inviting mountains, each with the promise of even grander views. With the purified enzyme, we learn about its catalytic activities and its responsiveness to regulatory molecules that raise or lower activity. Be- yond the catalytic and regulatory aspects, enzymes have a social face that dictates crucial interactions with other enzymes, nucleic acids, and mem- brane surfaces. To gain a perspective on the enzyme's contributions to the cellular economy, we must also identify the factors that induce or 4 METHODS IN ENZYMOLOGY [1] repress the genes responsible for producing the enzyme. Tracking a meta- bolic or biosynthetic enzyme uncovers marvelous intricacies by which a bacterial cell gears enzyme production precisely to its fluctuating needs. Popular interest now centers on understanding the growth and devel- opment of flies and worms, their cells and tissues. Many laboratories focus on the aberrations of cancer and hope that their studies will furnish insights into the normal patterns. Enormous efforts are also devoted to AIDS, both to the virus and its destructive action on the immune system. In these various studies, the effects of manipulating the cell's genome and the actions of viruses and agents are almost always monitored with intact cells and organisms. Rarely are attempts made to examine a stage in an overall process in a cell-free system. This reliance in current biological research on intact cells and organisms to fathom their chemistry is a modern version of the vitalism that befell Pasteur and that has permeated the attitudes of generations of biologists before and since. It baffles me that the utterly simple and proven enzymologic approach to solving basic problems in metabolism is so commonly ignored. The precept that discrete substances and their interactions must be under- stood before more complex phenomena can be explained is rooted in the history of biochemistry and should by now be utterly commensensical. Robert Koch, in identifying the causative agent of an infectious disease, taught us a century ago that we must first isolate the responsible microbe from all others. Organic chemists have known even longer that we must purify and crystallize a substance to prove its identity. More recently in history, the vitamin hunters found it futile to try to discover the metabolic and nutritional roles of vitamins without having isolated each in pure form. And so with enzymes it is only by purifying enzymes that we can clearly identify each of the molecular machines responsible for a discrete FIG. 1. Personalized license plate expressing a commitment to enzymology. [1] WHY PURIFY ENZYMES9. 5 metabolic operation. Convinced of this, one of my graduate students expressed it in a personalized license plate (Fig. 1). Acknowledgment This article borrows extensively from "For the Love of Enzymes: The Odyssey of a Biochemist," Harvard University Press, 1989. [2] GENERAL STRATEGIES AND CONSIDERATIONS 9 [2] Strategies and Considerations for Protein Purifications By STUART LINN The budding enzymologist is generally surprised by the time necessary to develop a protein purification procedure relative to the time required to accumulate information once the purified protein is available. While there is no magic formula for designing a protein purification, some forethought can help to expedite the tedious job of developing the purification scheme. This chapter is designed to point out some considerations to be under- taken prior to stepping up to the bench. Once at the bench, the subsequent chapters of this book as well as two other recent publications concerning enzyme purification 1,2 should serve as a guide. Preliminary Considerations What Is the Protein To Be Used For In these days of the biotechnology revolution, the required amount of purified protein may vary from a few micrograms needed for a cloning endeavor to several kilograms required for an industrial or pharmaceuti- cal application. Therefore, a very major consideration is the amount of material required. One should be aware of the scale-up ultimately ex- pected, and the final scheme should be appropriate for expansion to those levels. There are very real limitations to how far a procedure can be scaled up. These limitations are brought about not only by considerations of cost and availability of facilities, but also by physical constraints of such factors as chromatographic resin support capabilities and electro- phoresis heating factors. As outlined below, individual steps of the proce- dure should flow from high-capacity/low-cost techniques toward low- capacity/high-cost ones. Nonetheless, in some cases two procedures may be required: for example, one to obtain microgram quantities for cloning and a second to produce kilogram amounts of the cloned material. The protein chemist should remain flexible for adopting new procedures when such changes are warranted. Another consideration is whether the protein must be active (an en- zyme, a regulatory protein, or an antibody, for example), whether it must R. K. Scopes, "Protein Purification, Principles and Practice," 2nd Ed. Springer-Verlag, New York, 1987. 2 R. Burges, ed., "Protein Purification, Micro to Macro." Alan R. Liss, New York, 1987. Copyright © 1990 by Academic Press, Inc. METHODS IN ENZYMOLOGY, VOL. 182 All rights of reproduction in any form reserved. [...]... [2] worth the effort to carry out stability studies (e.g., heat inactivation or storage trials) in order to learn how to maintain a stable protein Two notes of caution: (1) optimal storage conditions change with purification; (2) optimal storage conditions need not relate to optimal conditions for activity Indeed, additions which stabilize a protein often inhibit it when added to activity mixes Of... the laboratory is the centrifuge The workhorse of the protein purification laboratory is the refrigerated high-speed centrifuge which attains speeds up to 20,000 rpm The usefulness of such a centrifuge is directly related to the presence in the laboratory of a wide variety of rotors and centrifuge tubes and bottles Rotors are available that hold as small a volume as a few milliliters per tube to ones... cost) In our labora- [3] SETTING UP A LABORATORY 23 tory, we have found that simple, open-top columns fitted with stoppers and syringe needles, or tubing, for fluid inlet and control, are satisfactory for most chromatographic procedures A dependable fraction collector is one of the most important pieces of equipment in the laboratory Failure of a fraction collector may result in the loss of several month's... when utilizing the protein interfering substances will have to be removed or "diluted out" during utilization of the protein In our experience, reducing agents are particularly effective with bacterial enzymes which derive from a reducing environment, whereas mammalian cell enzymes take kindly to surfactants and protease inhibitors Fungal proteins also respond to protease inhibitors Optimal pH and... Again, protein assay procedures can and often must be changed as the purification progresses What Should Be Added to the Buffers Once a purification scheme is developed, there is great resistance to modifying it, as modification requires laborious trial runs The usual response to " w h y is the protein suspended in x ? " is "if I leave it out, I don't know what will happen." The obvious lesson is to add... aside Metal chelators Sulfhydryl agents Ligands Protease inhibitors Examples KC1, NaCI, (NH4)2SO4 Deoxycholate Triton X-100 EDTA (ethylenediaminetetraacetic acid), EGTA [ethylene glycol bis(/3-aminoethylether) N,N'-tetraacetic acid] 2-Mercaptoethanol, dithiothreitol Mg2+, ATP, phosphate PMSF (phenylmethylsulfonyl fluoride), TPCK (N-tosyl-L-phenylalanine chloromethyl ketone), TLCK (N~-p-tosyl-Llysine chloromethyl... resin Nowadays, no protein purification laboratory is complete without the presence of gel electrophoresis equipment These items are used to monitor a purification procedure or for fractionation itself Generally, a vertical slab-gel apparatus with various-sized spacers and combs is satisfactory for most applications An electrophoresis power supply unit is also required If only one is to be purchased,... accompanied by greater rewards and indeed for protein chemistry as well [3] SETTING UP A LABORATORY 19 [3] S e t t i n g U p a L a b o r a t o r y By MURRAY P DEUTSCHER The aim of this chapter is to provide some general information on the basic equipment, chemicals, and supplies that should be present in any laboratory undertaking protein purification Details relevant to individual pieces of equipment, information... laboratory They are generally the least costly, used most frequently, required in largest numbers, and are the most essential It is natural in setting up a laboratory to focus on the large, expensive apparatus, but in practice, available funds should first go to ensuring an adequate supply of supporting materials (It obviously makes no sense to buy a sophisticated fraction collector, and not to have... in protein purification never has enough fraction collectors, columns, and gel electrophoresis apparatus Detection and Assay Requirements Probably the most important detection device in the laboratory is the spectrophotometer It can be used for determining protein concentrations, measuring the growth of bacterial cultures, as well as for a variety of enzymatic and colorimetric assays The spectrophotometer . for Protein Purifications By STUART LINN The budding enzymologist is generally surprised by the time necessary to develop a protein purification procedure relative to the time required to. inactivation or storage trials) in order to learn how to maintain a stable protein. Two notes of caution: (1) optimal storage conditions change with purification; (2) optimal storage conditions. Fractionation Requirements Protein purification means protein fractionation. What distinguishes a protein purification laboratory from the usual biochemistry or molecular biology laboratory is largely