Methods of Two-Dimensional Gel Electrophoresis for the Clinical Laboratory

Tracy, Russell P.; Currie, R M; Hill, Helene Z.; Young, Donald S.

doi:10.1016/b978-0-08-029815-3.50131-8

Cited by 10 publications

(5 citation statements)

References 14 publications

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“…This gradient gives the best resolution in the mass range of 100,000-10,000 daltons. To visualize small molecular weight proteins, a special gradient was designed, utilizing highly cross-linked, high acrylamide concentration gels polymerized in the presence of urea [26]. These gels contained gradients in acrylamide (15-25%), cross-linker (1:18.5-1:15) and urea (6-8 M).…”

Section: Sds-page Dimensionmentioning

confidence: 99%

Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

et al. 1984

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We have characterized the noncollagenous proteins of bovine bone using two high-resolution gel electrophoretic techniques. Proteins were extracted from bone tissue by extended dialysis against 0.5 M EDTA. In some cases, a preextraction was done in guanidine HCl. Bovine plasma was also examined to identify the proteins in bone that might also be present in blood. We have scored 160 major, reproducible spots on our standard preparation bone map. These comprise about 40 individual protein groups. There are many more minor spots present which puts the total number present over 200. Of these groups, 15 are not present on plasma maps. Bone proteins identified in this way include actin, bone Gla-protein, and osteonectin. The remainder are unknown. Bone Gla-protein is present in bovine bone in four isoelectric forms, pI = 3.95-4.50. Electroblotting analysis of EDTA and guanidine HCl extracted material failed to reveal any higher molecular weight immunologically reactive species. The plasma proteins found in bone include, but are not limited to albumin, apo A-I lipoprotein, IgG, IgM, transferrin, alpha-2-HS-glycoprotein, and hemoglobin. Extraction with guanidine HCl plus EDTA significantly enriches the yield for the nonplasma proteins but does not appear to extract any additional bone proteins.

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Section: Sds-page Dimensionmentioning

confidence: 99%

Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

et al. 1984

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“…In these studies, cytosol/microsomal, microsomal, or nuclear preparations were made from each type of cell population, and these were analyzed by 2D gel electrophoresis followed by silver staining. In this regard, the highly sensitive and semiquantitative silver staining procedure gives information about polypeptide/protein composition [13][14][15] and provides an excellent method to make comparative measurements on the composition of the amounts of specific proteins expressed in two related cell preparations. Table I1 reports that 1,169 unique spots were identified in silver stained 2D gels of cytosol/microsomal fractions.…”

Section: Resultsmentioning

confidence: 99%

“…The characteristics of the pH gradients were determined from pH readings on eluates of sections of several gels run in parallel. The second dimension employed gradients of 10-20% acrylamide prepared in 0.01 M Tris HCl buffer containing glycerol and SDS as described [13,14]. The DALT gels were run in the presence of Tris glycine buffer containing 3.5 mM SDS at 0.7 A.…”

Section: Two-dimensional Gel Electrophoresismentioning

confidence: 99%

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Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation

Wier

Scott

1987

J of Cellular Biochemistry

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The differentiation of murine mesenchymal stem cells occurs in nonterminal and terminal phases. In previous reports we established the characteristics of nonterminally differentiated cells and showed that transition from the nonterminal to the terminal state of differentiation can be induced by human plasma. We also showed that this transition is blocked by protein synthesis inhibitors and other pharmacological agents. In this paper, we have employed two-dimensional gel electrophoresis to evaluate changes in specific polypeptides that are induced when cells lose proliferative capacity associated with the terminal event in differentiation. Using silver staining procedures for analysis of electrophoretograms, we detected only seven major polypeptide differences between nonterminally differentiated and terminally differentiated cells. Six polypeptides were expressed only in preparations of terminally differentiated cells; these included two polypeptides identified in cytosolic fractions and four polypeptides identified in nuclear fractions. One polypeptide was also found to be selectively expressed only in nuclear fractions of nonterminally differentiated cells. Based on these observations we conclude that the loss of proliferative potential that occurs during the terminal event in mesenchymal stem cell differentiation is associated with changes in the composition of a limited number of specific polypeptides. We suggest that one or more of these polypeptides may be important in the regulation of cellular proliferation.

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“…The development of high-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) has enabled * Supported by NIH grant CA42522 Offprint requests to: Terry Maloney, Michigan Cancer Foundation, 110 E. Warten Ave, Detroit, MI 48201, USA the separation, based on the molecular mass and isoelectric point, of thousands of polypeptides from a single sample [15,24]. Whole-cell protein profiles from different organs are distinguishable with this assay [19].…”

Section: Lntroductionmentioning

confidence: 99%

Two-dimensional gel analysis of polypeptides from normal, preneoplastic and neoplastic mouse mammary tissues

Maloney

Wei

1990

Cancer Immunol Immunother

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High-resolution two-dimensional polyacrylamide gel electrophoresis (PAGE) was employed to reveal tumor-associated polypeptide changes, using the BALB/c C4 line mouse mammary model system, for which phenotypic and immunogenic alterations accompanying tumor progression are well defined. In the first set of experiments, polypeptide patterns from 20 micrograms whole tissue lysates of normal mammary gland, C4 preneoplastic hyperplastic alveolar nodule outgrowth (HAN) and spontaneous tumor from C4 HAN were compared. In order to normalize for differential cellularity and extracellular protein content in the whole tissues, our analysis included polypeptide patterns from serum, increased concentration of protein from whole normal mammary gland, and primary cultures of epithelial cells from normal gland, HAN and tumor. Using a computer-based image-analysis system, 90 polypeptides were identified in C4 tumor that were absent in C4 HAN, normal mammary gland and serum. None of the 90 polypeptides could be shown to represent a definite qualitative change in the protein composition of tumor epithelium as they were found to be either present in a higher concentration of protein from whole normal gland, or present in the primary epithelial culture from HAN, or absent in the primary epithelial culture from tumor. Conversely in the second set of experiments, when epithelial cultures were used as the starting point for comparisons to locate tumor-associated polypeptides, none of the 15 polypeptides that were present in cultures from three different tumors, and absent in the culture from normal mammary gland was specific to C4 tumor, as they were present in whole tissues of normal gland. Thus our experimental approach detected significant quantitative but no qualitative polypeptide changes in whole tumor tissue, or in tumor-derived epithelial cell cultures. This finding may reflect the limitations of the two-dimensional PAGE method, and warrants caution in the use of such gel analysis alone to identify tumor-associated proteins.

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Methods of Two-Dimensional Gel Electrophoresis for the Clinical Laboratory

Cited by 10 publications

References 14 publications

Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

Identification of the noncollagenous proteins of bovine bone by two-dimensional gel electrophoresis

Polypeptide changes associated with loss of proliferative potential during the terminal event in differentiation

Two-dimensional gel analysis of polypeptides from normal, preneoplastic and neoplastic mouse mammary tissues

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