24-well plate:
| 1 Tre:BACE2+FRET Substrate 2 | 2 Tre:miRG1,Hef1a:BACE1 (0 Dox) +FRET Substrate 1 | 3 Tre:miRG1,Hef1a:BACE1 (1000 Dox) +FRET Substrate 1 | 4 Assay Buffer 2 + FRET Substrate 2 | 5Assay Buffer 1 + FRET Substrate 1 | 6 HEK cells (no substrate) |
| 7 Purified BACE1 + FRET Substrate 1 | 8 Purified BACE1, BACE1 Inhibitor + FRET Substrate 1 | 9 Tre:miRG1,Hef1a:BACE1 (0 Dox), BACE1 Inhibitor +FRET Substrate 1 | |||
| 5 uM Reference Standard+ FRET 1 | 2.5 uM Reference Standard+ FRET 1 | 1.25 uM Reference Standard+ FRET 1 | 0.63 uM Reference Standard+ FRET 1 | 0.32 uM Reference Standard+ FRET 1 | 0.16 uM Reference Standard+ FRET 1 |
| 5 uM Reference Standard+ FRET 2 | 2.5 uM Reference Standard+ FRET 2 | 1.25 uM Reference Standard+ FRET 2 | 0.63 uM Reference Standard+ FRET 2 | 0.32 uM Reference Standard+ FRET 2 | 0.16 uM Reference Standard+ FRET 2 |
Make sure all components are at same temperature
store enzyme solutions on ice
Prepare Tissue:
4-5X lysis buffer to tissue sample ( could use assay buffer. Talk to Brian about homogenizing tissue samples, and amount of tissue)
incubate for 10 minutes on ice
centrifuge and collect supernatant
Prepare substrates:
for BACE2 - A:C 1:100