Associated Problems of Protein Electrophoresis, Staining and Densitometry*

ANNALS OF CLINICAL AND LABORATORY SCIENCE, Vol. 8, No. 5 Copyright © 1978, Institute for Clinical Science 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

Load more