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Assay Method Information |
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| | Immunofluorescence Resonance Energy Transfer (FRET) Assay |
| Description: | The FRET assay was performed essentially as described in Gruninger-Leitch et al., Journal of Biological Chemistry (2002) 277(7) 4687-93 (Substrate and inhibitor profile of BACE (beta-secretase) and comparison with other mammalian aspartic proteases). In summary, a peptide is designed that is cleaved by the protease. The peptide is labelled with dabcyl at the N terminus and Lucifer Yellow at the C-terminus, such that for an intact peptide the Lucifer Yellow fluorescence is quenched by the dabcyl. When the peptide is cut by BACE2, the quenching is removed and a fluorescent signal is generated. The assay was performed as described in Grueninger et al. 2002 at pH 4.5 using a substrate concentration of 5 μM. A FRET peptide based on the TMEM27 sequence was devised. dabcyl-QTLEFLKIPS-LucY (SEQ ID NO: 1). BACE2 had a high activity against this sequence, which is unrelated to the known APP-based substrates. Conversely, BACE1 had insignificant activity against this peptide. |
| Affinity data for this assay | |
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