Changes in protein expression during melanoma differentiation determined by computer analysis of 2-D gels.

Cytodifferentiation in many melanocytic cells is regulated through the adenylate cyclase-cAMP pathway. To analyse the molecular changes associated with this process we have compared the proteins produced by two closely related cell lines which, though derived from a single cell line, respond very differently to modulation of this signalling pathway. The human melanoma cell line DX3 shows little change in in vitro characteristics following treatment with cAMP elevating agents; in contrast the more malignant DX3 LT5.1 variant, derived from the DX3 parental line, shows pronounced dendrification, decreased proliferation and a reduction in metastatic capacity after similar treatment. The two cell lines were treated with phosphodiesterase inhibitors for 5 days and then processed for two-dimensional gel characterization using an immobilized pH gradient for the IEF dimension. Proteins were detected by silver staining the gels and protein intensities were digitized using a laser densitometer. Two-dimensional gel patterns were edited, matched and a melanoma protein database of 637 spots constructed using PDQUEST software on an Orion 1/05 computer. Eleven proteins were lost and four new proteins were detected in both cell lines following treatment. Twenty-two proteins were present in DX3 LT5.1 after treatment but not in untreated lines or treated DX3. These differentially expressed proteins may be associated with the observed changes in differentiation patterns and metastasis. Our results illustrate the resolving power of this technique and suggest potential applications to the study of cellular differentiation.

Stable storage conditions of Immobiline chemicals for isoelectric focusing.

Most of the problems connected with the use of the Immobiline chemicals (a set of six, non-amphoteric, acrylamido buffers having pK values in the pH 3.5-9.5 interval) can be attributed to the alkaline species (with pK values 6.2, 7.0, 8.5 and 9.3). These compounds, to varying degrees are subjected to two degradation pathways: (a) hydrolysis of the amido bond, producing free acrylic acid and a diamine, the latter unable to be incorporated into the polyacrylamide matrix; (b) spontaneous auto-polymerization, producing a number of oligomers up to n-mers, able to aggregate and precipitate large proteins. Storage of their water solutions as frozen aliquots, a method widely employed, only partially alleviates the problem. Addition of trace-amounts of inhibitors, as lately adopted by the manufacturer, could only reduce the problem of auto-polymerization, but not block the hydrolysis of the amido bond. A new solution has been found, which abolishes both phenomena: storage in n-propanol. As demonstrated by gas chromatography, HPLC analyses and two-dimensional separations of complex samples, storage in organic solvent completely abolishes both hydrolysis and auto-polymerization and allows production of highly reproducible focusing patterns.

Two-dimensional electrophoresis with immobilized pH gradients of leaf proteins from barley (Hordeum vulgare): method, reproducibility and genetic aspects.

Leaf proteins from 14 barley cultivars (Hordeum vulgare) were analyzed by two-dimensional electrophoresis with immobilized pH gradients (IPG 4-7 and IPG 6-10) in the first dimension. Highly reproducible two-dimensional patterns were obtained, owing to constant spot positions along the isoelectric focusing axis. A number of variety-specific protein spots were detected, allowing us to discriminate barley cultivars not only into main groups but into individual cultivars.