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Isolation of melanosomes from hair or feathers is difficult due to the need to break keratin structures using a strong chemical treatment. All recently published isolation approaches were examined in the isolation of melanosomes from human and dog hair. The best results were achieved from immediate separation of melanosomes by centrifugation after controlled NaOH hydrolysis of the hair. The crucial factor for successful separation is the choice of the optimal amount of time required for disintegration of the hair: if the time chosen is too long, melanosomes are damaged; if it is too short, contamination by residual keratin ensues. High temperatures must be avoided. Attempts to break hair structure by a number of milder reagents have failed.

Abstract: Normal melanosome biogenesis requires the association of structural proteins with tyrosinase. 3T3 Swiss fibroblasts transfected with mouse tyrosinase cDNA (line 13.4, clone c) are a unique system in which melanogenesis takes place in the absence of melanosomal structural proteins. Our study confirmed that transfected fibroblasts displayed tyrosinase activity and some of them produced pigment granules. In the absence of melanosomal structural proteins the granules failed both to show a typical ultrastructure and to undergo the usual melanosome ontogenesis. The differentiating agent – dimethyl sulfoxide- increased phaeomelanin production. Pigment was localized in membrane-bound vesicles which were identified as lysosomes by means of immunogold electron microscopy. Cell line 13.4 had higher levels of lysosomal enzymes (β-hexosaminidase, α-mannosidase) than both parental 3T3 cells and clone pKG4 (fibroblasts transfected with the G418 resistance plasmid). Melanosomal proteins act as scavengers of toxic products of melanogenesis, and our results suggest that in their absence cells may employ an alternative mechanism to sequester injurious products.