*Note: Protease inhibitors and BSA should be added to the buffer immediately prior to use.
BSA stock solution should be prepared at 100 mg/mL.
Note: All buffers, proteins, and tubes should be kept on ice until indicated. Assay volumes are 75 µL and are combined in 0.5 mL Eppendorf tubes. The table below Step 5 summarizes the reactions that should be set up in Steps 1-5.
Initial | Cleaved | Dil. | Mito. | Mito. | Final | Final |
Cleaved BID Conc. | BID | Buffer | Buffer | Vol. | Vol. | Conc. |
(nM) | (uL) | (uL) | (uL) | (uL) | (uL) | (nM) |
500 | 15 | 20 | 28 | 12 | 75 | 100 |
150 | 15 | 20 | 28 | 12 | 75 | 30 |
50 | 15 | 20 | 28 | 12 | 75 | 10 |
15 | 15 | 20 | 28 | 12 | 75 | 3 |
5 | 15 | 20 | 28 | 12 | 75 | 1 |
1.5 | 15 | 20 | 28 | 12 | 75 | 0.3 |
0.5 | 15 | 20 | 28 | 12 | 75 | 0.1 |
0.15 | 15 | 20 | 28 | 12 | 75 | 0.03 |
Controls | ||||||
Spontaneous Release | - | 35 | 28 | 12 | 75 | 0 |
Total Cytochrome c | - | 35 | 28 | 12 | 75 | 0 |
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