We examined sera from a normal population by two-dimensional gel electrophoresis, to establish the normal pattern of serum proteins and to investigate genetic polymorphisms. With such information in hand, specimens from patients with certain diseases may be readily evaluated. Towards this goal, we optimized the ISO-DALT system (Proc. Natl, Acad, Sci, USA 74: 5421--5425, 1977) for routine phenotyping of alpha 1-antitrypsin, haptoglobin, GC-globulin, alpha 2-HS-glycoprotein, and transferrin, as well as a previously unknown polymorphic protein. We examined the effects of aging the specimens for 2 h at room temperature (no changes) or at -20 degrees C for several months (small changes), as well as serum/plasma differences and the effect of protease inhibitors. Silver-stain methods were modified to allow simultaneous staining of 10 gels, with reasonably good reproducibility of stain intensity. We quantitated silver-stained gels by densitometry of photographic transparencies. Very small samples suffice with this stain (0.5 microL of serum or plasma), allowing the use of "finger-stick" methods instead of venipuncture, yet the patterns are better resolved and easier to read than those for 10-microL specimens processed on gels stained with Coomassie Blue. Our techniques for rapidly removing albumin and IgG allow the investigator to examine areas on the gel that ordinarily are obscured. The region of haptoglobin has been examined by using serum from an ahaptoglobinemic donor. Finally, we present an expanded "normal" map illustrating the composite protein pattern.
We modified the ISO-DALT two-dimensional gel electrophoresis system to allow the routine examination of serum specimens from patients with monoclonal gammopathies. This system, MC-Iso 1, is characterized by a broad pH gradient for resolving the basic immunoglobulin heavy and light chains. The increased resolution of basic proteins may be explained on theoretical grounds by an increase in voltage in this region of the cell. Ancillary techniques, such as those for albumin removal and pI assignment through use of charge standards, have also been implemented. The locations of immunoglobulin heavy chains have been confirmed by examination of over 250 serum samples as well as by "electro-blotting," with use of specific antisera. IgG subclass may also be predicted by location, but not with perfect accuracy. Differentiation of kappa and lambda light chains by relative mobility has been examined; the predictive value for correct identification of kappa chains is 83%, that for lambda chains 69%. Several unknown proteins have been observed in macroglobulinemia, related to mu heavy chain. Finally, we have determined that there is excellent correlation between non-denaturing isoelectric focusing and our system for pI assignment of light chains. This has importance due to reports of the potential importance of light-chain pI in the development of renal disease in patients with monoclonal gammopathies.
A two-dimensional gel "map" of the proteins in normal cells and transformed cells may well help establish the presence of putative tumor-specific protein antigens, which can be isolated and used as antigens. We so examined normal colon mucosa and colon adenocarcinoma, using a sensitive silver stain. Samples were promptly prepared after excision by rapidly trimming the tissue to a small, grossly homogeneous piece, which was frozen on solid CO2. Frozen histological sections and permanent mounts were also prepared for several areas, to document the cellular constitution and to grade the carcinoma. Sample preparation for electrophoresis is described. The DNA was pelleted by centrifugation (108 000 x g x 1.5 h). Little or no pellet was observed, indicating complete solubilization. Many of the tumors contained proteases, liberated during sample preparation, so protease inhibitors were included. A highly reproducible pattern was observed for normal mucosa. Comparison of the gels from paired samples with histological data allows many of the spots to be tentatively assigned to the different cell types present in the mucosa (epithelium, connective tissue, or muscularis mucosa) and, in most tumors analyzed, detection of a series of spots that are not detectable in the pattern for normal tissue. These apparently tumor-associated proteins may be of epithelial cell or stromal origin.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.