Materials And Methods

Materials and methods: In order to perform the experiment two forms of epidermal model were used. One was excised breast skin that was obtained from plastic surgery and the other was reconstructed human epidermis. The reconstructed human epidermis was generated by seeding epidermal cells on inert filter substrates and allowing the cells to be exposed to air in order for the cells to fully differentiate and develop a stratum corneum (which allows the reconstructed epidermis to survive UV irradiation at doses comparable to human skin). After irradiation of the skin samples and control samples under lamps for varying lengths of time the samples were lysed and then run on a 2D-PAGE and stained with silver or Coomassie G250 and then analysed. As the 2D-PAGE showed down regulation of two proteins (identified by Mass Spectrometry) Cofilin-1 and Destrin and a discrepancy in their pI from the theoretical the researchers thought that it could be due to phosphorylation of the proteins as it is known that this lowers the pI of proteins. To test this they digested the 2D-PAGE spots using Trypsin and then ran them through a Gallium (III) chromatography column as phosphorylated proteins stick to heavy metals like Gallium. Using Western Blot Analysis the researchers discovered that the decrease in the levels of phospho-cofilin-1 detected were due to de-phosphorylation of the protein and not degradation shown by the reduction in volume of phosphocofilin-1 but not a reduction in the overall levels of cofilin-1.