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 | 10 | 11 | 12 |
| 13 5 uM Reference Standard+ FRET 1 | 14 2.5 uM Reference Standard+ FRET 1 | 15 1.25 uM Reference Standard+ FRET 1 | 16 0.63 uM Reference Standard+ FRET 1 | 17 0.32 uM Reference Standard+ FRET 1 | 18 0.16 uM Reference Standard+ FRET 1 |
| 19 5 uM Reference Standard+ FRET 2 | 20 2.5 uM Reference Standard+ FRET 2 | 21 1.25 uM Reference Standard+ FRET 2 | 22 0.63 uM Reference Standard+ FRET 2 | 23 0.32 uM Reference Standard+ FRET 2 | 24 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 Control:
purified BACE1 : Assay buffer (1D) 1:250
Prepare substrates:
for BACE2 - component A:C in 1:100 ratio
for BACE1 component A: D in 1:100 ratio
put 50uL of tissue sample supernatant in each well. wait 10 minutes for everything to get to the same temperature.
put 50uL of appropriate substrate to each well
Measure 490/520nm every 5 minutes for 30-60 min. After add 50 uL of stop solution and measure again.