High resolution two-dimensional electrophoresis of proteins.

An improved procedure for staining of proteins following separation in polyacrylamide gels is described which utilizes the colloidal properties of Coomassie Brilliant Blue G-250 and R-250. The new method is based on addition of 20% v/v methanol and higher concentrations of ammonium sulfate to the staining solution previously described. The method combines the advantage of much shorter staining time with high sensitivity, a clear background not requiring destaining, stepwise staining, and stable fixation after staining. The method has been applied to staining of polyacrylamide gels after sodium dodecyl sulfate-electrophoresis and isoelectric focusing in carrier ampholyte-generated pH gradients.

Improved staining of proteins in polyacrylamide gels including isoelectric focusing gels with clear background at nanogram sensitivity using Coomassie Brilliant Blue G-250 and R-250.

(1996). Progress with Proteome Projects: Why all Proteins Expressed by a Genome Should be Identified and How To Do It. Biotechnology and Genetic Engineering Reviews: Vol. 13, No. 1, pp. 19-50.

Progress with Proteome Projects: Why all Proteins Expressed by a Genome Should be Identified and How To Do It

The protein-mapping method which combines isoelectric focusing in acrylamide gel and gel electrophoresis was previously used mainly for the separation of plant proteins and human serum proteins. We investigated with this technique soluble proteins of mouse tissues (whole embryos, the liver of fetal and adult mice, kidneys) and the proteins of mouse serum. The technique was tested under a number of different conditions to find those best for our purpose; they may represent some general improvements in the method. The protein patterns show high resolution and excellent reproducibility. About 275 spots were found for fetal liver, about 230 for whole embryos (day 14 p.c.) and about 100 for serum. The fact that a high number of protein spots can be evaluated by a single and comparatively simple experiment suggests that this method may be useful as an assay system for induced point mutations. The protein patterns demonstrated are compared and discgs of dominant lethal examinations after acute and subacute application of these three substances.

Protein mapping by combined isoelectric focusing and electrophoresis of mouse tissues. A novel approach to testing for induced point mutations in mammals.

Regulation of ferritin genes and protein

Isoelectric focusing in polyacrylamide gels.

Isoelectric focusing in gels

SMALL amounts of ferritin are found in serum in normal and pathological states1,2, usually at levels directly related to the amount of tissue storage iron3,4. This relationship, however, does not hold in many cancers where grossly elevated levels can occur without a corresponding increase in iron stores5–9. These latter findings suggest a possible use of ferritinaemia in cancer diagnosis but the value of this test has not been clearly established Such use is complicated by the multiple potential sources of the serum ferritin which could affect its type as well as its amount. Much of the ferritin in cancer sera may reflect increased iron stores arising from chronic anaemia or from transfusions in treated patients. Some may also come from nonspecific tissue damage. The high levels of ferritin synthesis in some cancer cells10 and the presence in serum of isoferritins characteristic of some cancer and foetal tissues11,12, however suggest that much of the ferritin may come directly from cancer cells. If so, selective quantitation of these isoferritins may provide specificity in possible serodiagnosis. The multiple isoferritins found in most human tissues seem to be hybrid molecules composed of different proportions of two subunit types13. One type predominates in liver and spleen isoferritins, and the other in the more acidic isoferritins in heart, tumours and HeLa cells14. Different isoferritin populations can be distinguished immunologically, apparently on the basis of their subunit composition14,15. Ferritin is usually assayed by immunological methods, with crystalline liver or spleen ferritins as immunising antigens and reference standards. These ferritins consist almost entirely of the liver-type subunit15. Consequently, most assays based on these ferritins have only a low reactivity for the more acidic ‘carcinofoetal’ isoferritins which may contain less than 20% of the liver-type subunit14,15. We have, therefore, tested a variety of cancer sera and compared the apparent levels of ferritin given by a standard ‘liver-type’ assay with levels given by an assay whose specificity is directed against the more acidic isoferritins15. We report here preliminary evidence that the latter type of assay may significantly improve the usefulness of ferritinaemia as a tumour marker.

Ferritinaemia in cancer.

Small-scale fractionation of proteins and nucleic acids by isoelectric focusing in polyacrylamide gels*

Summary. Ferritins from human liver, spleen, heart, pancreas and kidney were compared by electrophoresis and isoelectric focusing in polyacrylamide gels, by immunodiffusion against antisera to homologous and heterologous ferritin, and in some cases by their cyanogen bromide peptides. All ferritins appeared to consist of a single species on gel electrophoresis with the exception of heart ferritin which separated into two major components. Small differences in electrophoretic mobility were found in all tissue ferritins. By contrast, all tissue ferritins were found to consist of multiple forms when analysed by gel electrofocusing. At least five isoferritins were found in most tissues, several of which were common to most tissues. At least two were common to all tissues. Those ferritins which were most easily distinguishable electrophoretically, e.g. spleen and heart ferritin, showed the greatest differences on gel electrofocusing. The ferritin profile was characteristic of each organ and was reproducible both within individuals and between individual tissues. There were striking differences in the iron content of the various isoferritins within a tissue. Further, the iron content of isoferritins common to more than one tissue varied with the tissue of origin. Some isoferritins in several organs and all of the isoferritins in pancreas appeared to contain little, if any, iron. All five tissue ferritins contained antigenic determinants in common with liver ferritin. However, an additional antigenic determinant was found in liver ferritin which was not detectable in the ferritins from the other organs.

Human Isoferritins: Organ Specific Iron and Apoferritin Distribution

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