How are proteins detected on the Fluidity One?

Detection of proteins on the Fluidity One is achieved using an amine reactive dye based on ortho-phthalaldehyde (OPA) chemistry (Lee et al). This is only added after the diffusion step, meaning the sizing has already taken place and is not affected in any way by the dye. The reaction of OPA with amines is fluorogenic: fluorescence is only emitted by the reaction products (Simons and Johnson), while the unreacted OPA has minimal fluorescence and so affords low background fluorescence. Use of OPA removes the necessity for a washing step to remove unreacted dye and so prevents loss during purification and allows much more rapid measurement than pre-labelling. Fluorescence excitation on the Fluidity One is at 365 nm and emission at 440 nm. The high sensitivity allows detection down to concentrations of 10 µg/mL.

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