Part:BBa_K2027000:Experience

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Applications of BBa_K2027000

To characterize the construct's (L-lysine Alpha Oxidase) function, we induced a reaction between the purified protein samples (samples in equilibration buffer, wash buffer, and elution buffer) with racemic lysine. H2O2 is a side product of the reaction between lysine and L-lysine alpha oxidase. Following the reaction induction, we performed the Amplex Red hydrogen peroxide assay on the protein purification fractions. We had a standard curve of H2O2 of 0-10 uM H2O2 (before the addition of Amplex Red Working Solution). Based on the plate data, the elution buffer fraction displayed the most H2O2 activity compared to the other fractions. This was plausible since the elution buffer fraction contains the greatest and most purified amount of L-lysine alpha oxidase.

Below is a screenshot of the previously mentioned plate data for our induced reaction and H2O2 assay with the enzyme. Wells A1 through A11 are part of the standard curve. Well A1-A10 are respectively 1-10 uM H2O2. Well A11 is 0 uM H2O2 and contains only the 1X reaction buffer that is part of the kit. Row B contains reactions that have been quenched after 30 minutes. Row B contains reactions that were induced 5 minutes before the plate was read by the spectrophotometer. Wells B1-B4 and C1-C4 contain the lysine reaction with varying amounts of elution buffer-protein solution. Wells B5-B9 and C5-C9 contain the lysine reaction with varying amounts of wash buffer-protein solution. Wells B10-B12 and C10-C12 contain the lysine reaction with varying amounts of equilibration wash buffer-protein solution.

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