24-well plate:
| 1 Tre:BACE2+FRET Substrate 2 0 Dox | 2 Tre:miRG1,Hef1a:BACE1 (0 Dox) +FRET Substrate 1 | 3 Tre:miRG1,Hef1a:BACE1 (1000 Dox) +FRET Substrate 1 | 4 Tre:BACE2+FRET Substrate 2 1000 Dox | 5 Assay Buffer 1 + FRET Substrate 1 | 6 Assay Buffer 2 + FRET Substrate 2 | 7 Purified BACE1 + FRET Substrate 1 |
| 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 |
Assay Buffer 2 + FRET Substrate 2
Make sure all components are at same temperature
store enzyme solutions on ice
Prepare Tissue:
disassociate cells. count cells (so we use same amount each time). Dilute cells 0.5mL per 5-10 million cells in assay buffer.
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 (1F) : Assay buffer (1D) 1:250
50 uL total: 0.2 uL 1F : 49.8 uL
Prepare substrates:
for BACE2 - component 2A:2C in 1:100 ratio
8x50uL = 400uL: 4uL 2A : 396 uL 2C
for BACE1 component 1A: 1D in 1:100 ratio
12x50uL = 600uL: 6uL 1A : 594uL 1D
Prepare Reference standards:
DIlute reference standard 1:100 in assay buffer. Make subsequent 1:2 dilutions to make reference ladder. Do the same for BACE2 substrate?
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 E and measure again.