Associated Problems of Protein Electrophoresis, Staining and Densitometry*

BENNIE ZAK, Ph.D.,f EUGENE S. BAGINSKI, Ph.D.,* and EMANUEL EPSTEIN, Ph.D.§

Departments of Clinical Pathology: t Wayne State University School of Medicine and Detroit General Hospital, Detroit, MI 48201 f St. Joseph Mercy Hospital, Pontiac, Ml 48053 § William Beaumont Hospital, Royal Oak, MI 48072

ABSTRACT The process of electrophoresis, a separation phenomenon, is mistakenly understood to include the sequential processes ancillary to analyte resolu tion, that is, staining and quantification, where the latter could be elution followed by photometry or integrating-calculating-densitometry. The theories involved in electrophoresis itself are well worked OuFand equally well understood but the problems which are associated with separation, chemical reaction to generate a chromogen and quantification, perhaps partly forgotten and perhaps partly ignored, are taken up and described here. They include albumin trail, resolution, unequivalent staining, prestaining and the densitometry problems associated with band widths, opacity effects and polychromaticities.

Introduction and perfected of the three phases in terms of accomplishing its purpose with the Any discussion of protein electro least problems, that is to separate proteins phoresis should include electrophoresis into several well resolved zones by the itself, and the sequential phases as simplest means possible. sociated with the process, initially, fixa The pattern from any electrophoresis tion staining and finally, quantification of process may contain in each zone several the stained zones. Electrophoresis, the protein constituents, including iso separating and resolving stage of the pro enzymes, lipoproteins, hemoglobins and cedure, is perhaps the more controlled haptoglobins among other potential ana lytes obtained by the type of fixation- staining used to visualize the individuals * Supported in part by a Grant-in-Aid from the Detroit General Hospital Research Corporation and of each family of constituents to be con N.I.H. Grant Number 05384-16. sidered. The serum protein electro 385 0091-7370/78/0900-0385 $01.80 © Institute for Clinical Science, Inc. 386 ZAK, BAGINSKI AND EPSTEIN phoretic pattern commonly obtained in Although great improvements have clinical laboratories usually consists of been made in the separation of proteins, five to six zones. Although each zone is especially when filter paper was replaced highly heterogeneous, the procedure is as the anticonvection medium by gels still quite useful as a tool in diagnosis. The such as cellulose acetate or agarose, the art number of zones resolved can be in of staining and quantification of protein creased considerably if a molecular sieve has changed very little. Much of the sci support, such as polyacrylamide or starch ence pertaining to stain-protein charac gel, is used as the anticonvection medium teristics originated in. work using filter in place of the more commonly used cel paper as the anticonvention, staining and lulose acetate or agarose gel. quantification medium. It is perhaps pos sible to extrapolate from some of the ap However, the clinical interpretation and, therefore, the need for these more plications using one type of medium to complex patterns of proteins has not been that of another type of medium, but it may well established except perhaps in typ be difficult to make a quantitative com parison between studies concerning ing.18 Unless this superior technology of separation results in better interpretations albumin-globulin relationships obtained with the large number of individual frac by separations in different media. tions, a reproducible separation of serum As an example, albumin trailing (tail proteins into fewer well resolved bands ing) is a dominant phenomenon in paper appears to suffice for the time being. It is electrophoresis,4 but not in agarose in the next two procedural phases follow electrophoresis. Therefore, it is only a ing separation in which one must look for secondary purpose of this paper to enum the significant problems of the entire erate and discuss some of the problems process, because it is in staining and quan associated with the separation of proteins tification in which important difficulties and the subsequent fixation-staining are encountered. processes. However, the primary and most important purpose is the delineation The staining and quantification phases of several problems of densitometry of the entire electrophoretic procedure which are not as well realized as those of should probably be discussed together the first two phases. Relating the previous because there is a close interrelationship stages of the procedure to the final meas between several factors, perhaps among urement, densitometry, may help in the others, such as instrument measuring understanding of why a spectrophoto- capabilities, dye-protein complexes, metric process usually involving solid spectral characteristics of these com color measurement can achieve results plexes in various media, liquid or dry, the which are different than and less accurate geometry of measurement and the con than another form of spectrophotometry, centration of proteins in each zone.4'7'19' elution followed by absorptiometry.32 20, 21, 25.34 Since the amount of dye-uptake by the protein is commonly related to pro The need to understand why all vari tein concentration by some quantification ables of the three dimensional measure process such as measurement by direct ment of densitometry (length, width and densitometry or elution followed by depth, the latter manifested by absor spectrophotometry, inherent pitfalls con bance) must be controlled will be de nected with protein dye-binding and the scribed along with the effect that an subsequent measurements of dried strips opaque medium produces in the process or eluates must be recognized before pos of making such a volumetric measure itive quantitative interpretations from the ment. A final point to mention is the results obtained are attempted.7 polychromatic light effect, since there PROBLEMS OF PROTEIN ELECTROPHORESIS, STAINING AND DENSITOMETRY 387 have been instances of the use of extremes usually follows. A number of analytical in light source in densitometry varying problems are associated with this process. from white light to monochromatic Several of these might be described in the light.4,25 following manner:

1. Staining of proteins may show that a Principles high concentration protein is under The principles involved in the protein stained, perhaps as a penetration effect, quantification process are necessarily while the low concentration protein may complex as would be expected of a mul- be completely stained. In this cir tiphasic procedure involving an electro cumstance, the high concentration pro phoretic separation, fixing-colorization tein would be underestimated and, in a step and, finally, an evaluation either by relative sense, the low concentration pro elution-colorimetry or integrating densi tein would be overestimated.12 tometry on liquid or solid samples. 2. Unlike proteins showing variations With reference to the first phase, in the number of binding sites would react electrophoresis is the migration of any diversely with different stains making re kind of charged high molecular weight so lationships nonlinear and empirical un lutes or particles in an electric field in a less corrective factors could be deter buffered medium, whereas the term mined. The latter might be difficult to iontophoresis is usually confined to de calculate because the individual zones scribing the migration of small ions. When are heterogeneous.23,33 an anti-convective support is used as the 3. Proteins vary in concentration across means of confining the buffer, the term wide ranges and show quotients of30:l to zone electrophoresis is applied to the pro 50:1 (HbA:HbA2). Staining under these cedure. The anticonvective support can conditions usually leads to a low protein be paper, cellulose acetate, agarose, agar, measured with less accuracy than the high polyacrylamide, starch or others. protein, if the latter is measured opti In this phase, buffered solution, usually mally. Conversely, if the low protein is at a pH around 8.6 to 9.0 and optimally measured optimally, the high protein is distant from the serum protein isoelectric measured with less surety.32 points, is fixed by an anticonvective sup 4. In keeping with the notion of the port such as paper, acrylamide, starch, cel previous statements, even if the high and lulose acetate or agarose and made part of low concentrations of proteins could be a circuit containing a DC power supply. stained uniformly, the measurement of The sample is applied to the support- the stained material might be non-linear buffer system in which the proteins re owing to deviation from Beer’s main negatively charged and voltage is law.19,20,21,31 applied. After a suitable predetermined 5. Some staining techniques utilizing time during which the differently charged tetrazolium salts, especially in cellulose proteins separate, electrophoresis is ter acetate procedures, must be scanned minated. against an uncleared and opaque background, a circumstance where blank ing is not easy. Unless some kind of empir Staining ical relationship is established in the In the staining phase, the separated pro calibration process, or unless the relative teins are simultaneously fixed to prevent absorbances are low enough to be in the further diffusion and are reacted chemi more linear area of measurement, this cally. Then, if cellulose acetate is the technique would present a difficult mate medium, a transparency or clearing step rial for the quantification phase.9 388 ZAK, BAGINSKI AND EPSTEIN

Quantification as with cellulose acetate, then only the most general directions for their separa Lastly, the concentrations of the several tion are feasible. Therefore, reagents and stained proteins are determined by meas procedures will not be described here for urement with an integrating, automatic the separation of proteins and their sub scanning densitometer which also con sequent staining. The densitometric as tains a minicomputer for all required cal pect of the triphasic system for determin culations such as those of relative or abso ing protein concentration of separated lute values of the analytes present. This zones following staining involves only scanning of the separated proteins is photometric measurements. The latter are carried out with a specially designed usually made in conjunction with simul spectrophotometer (continuous or a- taneous calculations based on those bridged across whose slit mechanism the measurements for the purpose of deter zones are passed at a uniform speed. The mination of relative values and/or sub zones are, as previously described, usu sequent conversion to absolute concen ally stained and dried into their anti- trations. convective medium such as paper, cel When both area and absorbance for lulose acetate or agarose before the final each zone are considered, the meas quantification step is carried out. With urements of each zone can be seen to in other less used clinical laboratory media volve the volume of each zone. The need such as polyacrylamide gel, the densi for narrow band photometers capable of tometry is carried out in the clear liquid linearly measuring across quite varied in gel using special holders. tensities of stained zones is obviously a Several leaders in the field suggest that useful requirement in densitometry. the quantification step could for the most Because so many instruments are cur part be eliminated and replaced by rently used for the determination of the specific protein determinations based on zone value by transmitted light, reflec the qualitative findings.15,17,23 Most clini tance or fluorescence, it would seem to be cal laboratories, however, still find densi unnecessary to describe the directions tometry or to a lesser extent, elution fol that the manufacturers all provide for each lowed by photometry, to be valuable aids instrument. Therefore, only the general in the solution of diagnostic problems. description described under Principles is Simpler quantitative systems than the all that is required here, just as was previ serum proteins, as for example HbA:HbA2 ously stated for separation and visual where two separated constituents may ization. need to be distinguished and compared to Although, not a part of densitometry it each other rather than the five to six zones self, some measure of the total concentra of serum proteins, have been indicted as tion of the separated mixture must be resulting in too high a percentage for known and therefore determined, if any HbA2 when densitometry is the quantifi thing more than the relative concen cation device.32 In that circumstance, trations of the separated analytes is re either elution or microcolumn chromatog quired. If the absolute concentrations of raphy is suggested as a more accurate al the individual proteins are needed, it ternative.31 becomes necessary to determine the con centrations of total proteins by biuret re Materials and Methods action, spectrophotometry, index of re fraction or whatever analytical device is When the large number of apparatuses deemed applicable to the separated zones. available for even one method of electro They could be proteins, enzymes, lipo phoresis of proteins are considered, such protein lipids, etc.31 PROBLEMS OF PROTEIN ELECTROPHORESIS, STAINING AND DENSITOMETRY 389 Discussion and Results three phases, providing that the several variables are considered carefully and A brief review of the variables in con used judiciously. ventional electrophoresis that must be Once the proteins are separated into controlled to enable precise resolution of groups, referred to as zones, and fixed and protein zones includes several separatory stained, there is the possibility of using conditions. Those factors which affect cel these patterns qualitatively followed by lulose acetate electrophoresis, aside from specific protein reactions for individual the physical properties of the protein, are proteins where abnormalities are obvious as follows: or it is necessary to quantify them. In the 1. The buffer characteristics such as staining processes, a denaturation step is pH, composition of the buffer and its at needed to immobilize the proteins and fix tendant ionic strength.4 the protein-dye complex into the medium. 2. High ionic strength decreases migra Excess dye is then removed by washing tion while heat effects may also decrease the gel with an appropriate wash solution migration. which should maintain the bound stain 3. The buffer imposes a charge on the and the protein without loss of either. proteins as a function of pH, and the latter The residual background may depend is selected as a function of the isoelectric on the number of washes applied and the points of the proteins. effectiveness of the washing solution. 4. Buffer concentration affects the reso Cellulose acetate, when dried, forms a lution characteristics of electrophoresis10 thin layer which is naturally opaque and inasmuch as the kind of compound used in needs to be cleared, a process with vari the buffer defines interaction with pro able degrees of success since some stains teins, heat effects and mobility of the pro are not compatible with known clearing teins. treatments.3 5. A compromise must be made in the Agarose gel, on the other hand, is usu ionic strength of the buffer because al ally prepared on a thin, clear plastic or though higher ionic strength permits bet glass plate and upon drying forms a thin ter resolution in theory, it also causes a transparent film with the protein zones heat effect in practice which can slow mi thus firmly attached to the solid surface.2,3 gration, so the lowest ionic strength which This technique allows one to handle a allows good resolution2 is usually the best strip or plate easily for further processing compromise. by direct densitometry or elution. In the 6. Other factors include the electric case of polyacrylamide gel, the proteins field and its relationship between voltage, are fixed and stained while the medium current and time,2 electroendosmosis and retains its liquid. Excess dye is sometimes the principle of the wick effect.3 removed by electrophoretic destaining, 7. The physical nature of the support that is, the gel is again subjected to medium can effect migration ranging from electrophoresis in a. fixing medium, which the minimal effort of dilute gels28 to the enables the free dye to migrate away from tortuous path because of the fibers of pa the gel while the protein-dye complex per16 or the sieving effect of controlled remains stationary.5,22,24- pore gels.5,24 It is particularly important here that 8. The physical properties of the pro complete denaturation of all proteins is tein molecules themselves affect the sep accomplished before electrophoretic de aration of proteins, their staining and ul staining is applied, otherwise unfixed timately, therefore, their measurement. remnant amounts of protein may be sub In summation, it might be stated that jected to migration and lost.6 Also, if the separation appears to be the best of the proteins are not insolubilized promptly, 390 ZAK, BAGINSKI AND EPSTEIN some proteins are reported to diffuse out Critical studies concerned with the of the residue.27 Some of the commonly characteristics of different dyes employed used fixatives such as acetic, sulfosalicylic in staining of electrophoretically sepa or trichloroacetic acids, dehydrating rated serum proteins were carried out by agents such as methanol or ethanol or a Luxton and others.12,19,20,21,22 Many of the combination of the acids and dehydrating problems associated with the staining agents may not be totally effective in process were described, but several con completely denaturing all proteins.1 Some siderations of the quantification process assert that picric acid13'18 or uranyl acetate need further emphasis. Inasmuch as this are most effective.1 last phase of the total procedure appears to Quantitative analysis of separated pro have had somewhat less coverage in the tein fractions is usually based on spectro clinical chemistry literature, most of the photometry following dye-binding. An remaining discussion will be concerned assumption is often made here that the with two important problems of densi uptake is the same on a weight basis for all tometry, and these are the effects of types of proteins. This, however, may not medium opacity and slit geometry34 on always be the case. For example, it was measurement accuracy. reported that albumin binds considerably The effect of opacity leads to distortions more Ponceau S dye than do the globu which are described by some as a lins.19'21 A similar difference was also con phenomenon involving multiple internal firmed for Ponceau S14 while variation in reflections. This has also been called a Amido Black uptake has been reported hyperchromic effect for an opaque among globulins.21 Furthermore, dye medium.22 However, a true hyperchromic uptake for presumably the same class of effect would be one which resulted in an protein differs between species. For ex increase in spectral band intensity, a ample, human albumin binds more Bro- property not really encountered in this mocresol Green dye than does bovine circumstance. If multiple internal reflec album in.29,30 tions are the true cause of the effect, it All this should not be surprising in view must be a factor that results in increased of the fact that proteins are highly absorbance values owing to an effective heterogeneous entities which differ in the lengthening of the light path. Therefore, if arrangement and type of reaction sites this were described as a Lambert effect, it which are responsible for the stain- would seem to offer an explanation which binding. It would be ideal to find a vis would be more consistant with the con ualizing chemical with which different cept of multiple internal reflections. proteins would exhibit the same reactive Anything that could cause the light path capacity on a weight basis. Originally, to be elongated, such as a material with Bromophenol Blue and Amido Black were opaque fibers, which would obviously re the two most common dyes used follow sult in increased absorption by the ing protein electrophoresis. Currently, chromogenic material would be subject to Ponceau S seems to be more popular as a Lambert’s law in the manner described. protein stain following electrophoresis in However, corrective blanking to over cellulose acetate or agarose gel. However, come this effect cannot be achieved here as already mentioned, albumin appears to by any simple means because th'e un have a higher binding capacity for that dye stained areas do not provide similar ab than globulins.19’20,21 If albumin binds sorption by the chromogen of the travers more of the dye than do globulins as is ing light beam. claimed,19,20,21 it would appear that the Although it is a known phenomenon use of Ponceau S stain should overesti that the degree of opacity of a medium mate the albumin and underestimate the affects densitometric measurements,9,26 it globulins. is still a medium of choice for certain PROBLEMS OF PROTEIN ELECTROPHORESIS, STAINING AND DENSITOMETRY 391 commonly used systems where reaction to cleared membrane, but for the second provide measureable chromogens is ac scan the cleared membrane was backed complished through reduction of tetra- with an uncleared piece of cellulose ace zolium compounds by coenzymes. Two tate membrane. The results are shown in common examples are in the separation of figure 2. In this case, the two patterns ap lactate dehydrogenases or creatine pear quite similar with no distortion appa kinases or in most commercial paper rent even when the transparent pattern electrophoresis systems. When the anti had the opaque backing. convection medium is cellulose acetate, The explanation lies in length of light clearing is not attempted and meas path, for in this case the light path is the urements are carried out in the presence same for both scans and the opaque of the opaque background.3 background is easily corrected. The sa The assumption is also made that since lient point is that it made a difference in the background is subtracted in the blank measurement whether the absorbing ing process, then the effect of the medium species were within the fibers or outside on the chromogens representing isoen of the fibers, for in the geometry of meas zymes will be corrected. That would be urement, a Lambert effect would be af true as previously stated if the light path fected by this dimension of the measure were not elongated by these multiple ment. internal reflections which result in greater A final experiment enforcing the find absorbance for the longest light paths by ings of figures 1 and 2 was carried out the colored species, a species not present using an optical wedge specifically de in the blank area, and a nonlinear signed to calibrate the scanning instru “hyperchromic-like” effect which might ment. These results are shown in figure 3. have to be corrected by means of the The upper scan shows the wedge values Kubelka-Munk9 version of the Beer- which should be in the ratio of Lambert law as derived for use in opaque 100:80:60:40:20 and the areas and shapes media. An alternative approach would be appear concordant with manufacturer’s empirical calibration by means of known specifications.3 When an uncleared cel concentrations of standards. lulose acetate back was attached to the Several experiments to explain this wedge, the lower scan was obtained. The phenomenon were carried out. In one, de rounding of the corners signals that some scribed by the densitometric tracing of thing is different for this scan, but there is figure 1, serum samples were electro- no severe distortion as there was with the phoresed in duplicate sets and then proteins of figure 1, again indicating that it stained with Ponceau S in a prescribed makes a difference if the absorbing mate manner.3 The patterns were then divided rial is within or outside of the fibers that into two sets where one was cleared and could cause light to engage in the the other left opaque. The scans of the phenomenon of multiple internal reflec dense background samples were mark tions, a Lambert effect. edly increased and distorted when com A preliminary study has already been pared to the transparent patterns. An described in which it was postulated that example of these distortions is shown in inaccurate relative areas could be caused the figure for one pattern in which this by a masking slit mechanism which under distortion effect is quite visible. There estimated major bands.7-34 The concept was no correspondence in relative values was based on the idea that using a masking obtained for any of the serums of the two slit smaller than the smallest band could sets. not be effective because all bands larger In a second experiment, a transparent than the smallest band would be under example of a myeloma serum separation estimated in proportion to their size ratios was used. It was scanned twice, once as a to those smallest bands. In effect, one di 392 ZAK, BAGINSKI AND EPSTEIN

F ig u re 1. Densitom etry of simultaneously separated serum protein patterns where one pat tern is cleared (Trans parent) and one is left opaque.

mension of a three dimensional meas values are later determined using the con urement is fixed by slit geometry, where centrations of the total protein present, it as it can be a variable which can result in is the latter determination from which mistaken relative calculations. subsequent calculated values may be dis In electrophoresis as it is performed in torted. Problems perhaps not unlike those the clinical laboratory, severely turbid of densitometry of an opaque medium serums are sometimes encountered. In may cause an apparent hyperchromic ef the electrophoresis procedure followed fect which may distort absorbances of by staining and densitometry, there may blank versus the sample as a Lambert ef be problems associated with the facts of fect. the lipids’ presence, but little appears to The quantitative data obtained after the be done about it in the measurement electrophoresis of serum proteins has process. However, since only relative been completed are often used as a values are obtained from which absolute yardstick for determining whether or not PROBLEMS OF PROTEIN ELECTROPHORESIS, STAINING AND DENSITOMETRY 393

: ; : l-L :■ n-1? M— - 44rH- -H : i'H N O f . : • : ; : ; : ; r i_t. ‘ Hr i! i : t : ;

F ig u r e 2. Densitom etry of a cleared pattern of myeloma serum (Transparent) versus the densitometry of the same pattern backed by an uncleared cellu lose acetate membrane (C.A. Backed).

other analytical procedures for determin tion of a spectrophotometric measure ing specific proteins are accurate. In order ment such as width of a uniform spot, for this to be true, certain factors would length of a uniform spot and the absor have had to be established for the triad of bance through a portion of that uniform sequential analytical steps of electro spot which could accurately convert the phoresis, dye-binding reaction and densi whole measurement to correct volume re tometry. Such factors would have had to lationships for the separated proteins. The include good if not perfect separation of problem appears to be complicated by the the proteins, uniform chemical reaction following facts: on a weight basis and densitometry. The 1. Albumin trail in some systems (pa densitometry did not preclude any por per) creates a condition in which the al- 394 ZAK, BAGINSKI AND EPSTEIN

TRANSPARENT BECKMAN DENSITOMETER STANDARD "

üiSiiRüiiilüü

C.A. BACKED

üiiMiliiilliüillHllllIltlüil! ...... li ìli i!l H i l Ifiìil iia a a ia 1!; ra i, ili m i

Figure 3. Densitometry of a Beckman optical wedge used as a standard (Transparent) versus the densi tometry of the same wedge backed by an uncleared cellulose acetate membrane (C.A. Backed). bumin concentration is decreased in the the excess dye ran ahead of albumin liter final analysis by the quantity bound to the ally into discard. During the fixation and anticonvective medium. dye-binding process, other stains, such as 2. Owing to the latter phenomenon, Amido Black may be used for all of the alpha and beta globulins are increased proteins. This results in a distortion of since they superimpose the trail while measurable absorbances for the indi gamma globulins under endosmotic in vidual proteins since the marker dye is fluence are neither high nor low, for their still bound to albumin but to no other pro movement is away from the trial area.4 teins, while the second dye is bound to all The different proteins can react differ of them as a function of the binding sites of ently from each other on a weight basis all of those proteins for that dye." with protein stains.19,20,21 Critical observa Even if the staining dye binds uni tion of electrophoretic patterns indicates formly on a weight basis, there could be that the albumin and the other proteins considerably perturbation of the final are not uniform in dimension along the measurement. In considering that albu axes perpendicular to the scan path. min may have the largest number of bind For many years, some analysts have ing sites for either pre-stain or Ponceau S, added stain, usually Bromphenol Blue, to the empirical results attained from meas serum which then acts as an albumin urements on a weight basis which are al marker at alkaline pH. By that means, the ready quantitatively unrelated are now prestained albumin could be followed worsened by this marker process. Obvi during the electrophoretic process while ously, if these facts are considered, the PROBLEMS OF PROTEIN ELECTROPHORESIS, STAINING AND DENSITOMETRY 395 marker dye should be the same dye as the 16. Ku nkel, H. G. and Tiselius, A.: Electrophor staining dye if this distorting phenome esis of proteins on filter paper. J. Gen. Physiol. 35:89-118, 1951. non is to be avoided. 17. La u r e ll , C. B.: Electrophoresis, specific pro tein assays, or both in measurement of plasma proteins. Clin. Chem. i9:99-102, 1973. 18. LOZSA, A.: Uranyl acetate as an excellent fixa tive for lipoproteins after electrophoresis on agarose gel. Clin. Chim. Acta 53:43-49, 1974. References 19. Luxton, G. C.: Serum protein electrophoresis. Evaluation and modification of the Micro Zone 1. ALBERT-RECHT, F.: Quantitation of plasma pro system. Part I. ??? Canad. J. Med. Tech. 26:4- teins on cellulose acetate strips. Clin. Chim. 17, 1968. Acta 4:627-638, 1959. 20. Luxton, G. C.: Serum protein electrophoresis. 2. B a r t l e t t , R. C.: Rapid cellulose acetate Evaluation and modification of the Micro Zone electrophoresis: I. Serum proteins. Clin. Chem. system. Part II. Ponceau S. Canad. J. Med. 9:317-324, 1963. Tech. 26:55-70, 1968. 3. B e c k m a n M ic r o z o n e E lectrophoresis 21. Luxton, G. C.: Serum protein electrophoresis. M a n u a l . Gebott, M ., ed. Beckman Instruments Evaluation and modification of the Micro Zone Inc., Fullerton, CA, 1975. system. Part III. Possible dye substitutes for 4. Bl o c k , R. J., D u r r u m , B. L., and Z w e ig , G.: A Ponceau S. Canad. J. Med. Tech. 26:83-101, Manual of Paper Chromatography and Paper 1968. Electrophoresis. New York, Academic Press, 22. M a t SON, C. F.: Polyacrylamide gel electro Inc., pp. 349-390, 1955. phoresis. A simple system using gel columns. 5. D a v is , B. J.: Disc electrophoresis. II. Method Anal. Biochem. J3:294-304, 1965. and application to serum proteins. Ann. N. Y. 23. Neren b erg , S. T.: Electrophoretic Screening Acad. Sci. 121:404-427, 1964. Procedures. Philadelphia, Lea and Febiger, 6. D i e z e l , Kopperschlager , G., and H o f f 1973. m a n n , N. E.: An improved procedure for pro 24. O rnstein, L.: Disc electrophoresis. I. Back tein staining in polyacrylamide gels with a new ground and theory. Ann. N. Y. Acad. Sci. type of Coomassie brilliant blue. Anal. 121:321-349, 1964. Biochem. 48:617-620, 1972. 25. P E E T E R S , H.: Paper electrophoresis: principles 7. E p s t e i n , E ., Ba g in s k i, E . S., and Z a k , B.: Pro and techniques. Advances in Clinical Chemis teins: zone separation, visualization and quan try, vol. 2. Sobotka, H. and Stewart, C. P ., eds. tification. Seminar on Proteins and Protein- New York, Academic Press, pp. 4-134, 1959. opathies. Sunderman, F. W., ed. Philadelphia, 26. Q uantitative Th in L a y er C h r o m a t o g Institute for Clinical Science, pp. 6-21, 1977. r a p h y : A Symposium. Vitatron N V, Holland, 8. E p s t e i n , E . and Z a k , B.: Separation of hapto 1971. globins by disc electrophoresis. Ann. Clin. Lab. 27. Racusen, D.: Stoichiometry ofthe Amido Black Sci. 2:191-197, 1972. reaction with proteins. Anal. Biochem. 52:96- 9. GOLDMAN, J. and G o o d a l l , R. R.: Quantitative 101, 1973. analysis on thin layer chromatograms: a theory 28. Ressler, N. and Zak, B.: Electrophoresis in a for light absorption methods with experimental fluid film. Clin. Chim. Acta 1:393-400, 1956. details. J. Chromatog. 32:24-42, 1968. 29. Ro dk ey, F. L.: Binding of bromocresol green 10. G r a n t , G . H. and Ka c h m a r , J. F.: The proteins by human serum albumin. Arch. Biochem. of body fluids. Fundamentals of Clinical Biophys. i 08:510-513, 1964. Chemistry. Tietz, N. W., ed. Philadelphia, W. B. 30. RODKEY, F. L.: Direct spectrophotometric Saunders Co., pp. 298-400, 1976. determination of albumin in human serum. 11. H a ttin g h , J., COETZEE, N., and ROSS, F. P.: Clin. Chem. 11:478-487, 1965. Erroneous densitometric albumin.estimation as 31. S c h m i d t , R. M. and BROSIUS, E. M.: Basic Lab a result of prestaining with certain dyes. Anal. oratory Methods of Hemoglobinopathy Selec Biochem. 80:635-638, 1977. tion, 8th ed. Washington, HEW Publ. No. 12. H en ry , R. J., G o l u b , O. J., and So b e l , C.: (CDC) 76-8266, 1976. Some of the variables involved in the fractiona 32. Sc h m id t , R. M., Ru c k n a g l e , D. L., and tion of serum proteins by paper electrophoresis. Ne c h e l e S, T. F.: Comparison of methodol Clin. Chem. 3:49-64, 1957. ogies for thallasemia screening by Hb A2 13. J o h a n s s o n , B. G.: Agarose gel electrophoresis. quantitation. J. Lab. Clin. Med. 86:873-882, Scand. J. Clin. Lab. Invest. 29:7-19, 1972. 1975. 14. Ke y s e r , J. W. and St e p h e n s , B. T.: Estimation 33. Sunderm an, F. W., Jr . and Sunderm an , F. W.: of serum albumin: A comparison of three Studies on the serum proteins. IV. The dye- methods. Clin. Chem. 8:526-529, 1962. binding of purified serum proteins separated by 15. Ko h n , J.: Routine serum protein analysis: continuous-flow electrophoresis. Clin. Chem. trends, facts and fallacies. Protides of the 5:171-185, 1959. Biological Fluids—23rd Colloquium. Peeters, 34. Zak, B., Epstein , E., and Watkins, R.: Postu H., ed., Oxford and New York, Pergamon Press, lated flaw in densitometry. Microchem. J., in pp. 325-331, 1976. press.