Matt Gethers/CRI, Thailand/Labwork/Binding Assay/8.8.08 Run Notes
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8.8.08 Binding Assay Run
Volumes and Concentrations of Constituents | Volume | Final Concentration | Volume removed for next dilution | Final Volume |
8.44 μl of stock protein (assume 90 μM monomer) in 29.55 μl of 1x buffer (+DTT) | 38 μl | 20 μM | 18 μl | 20 μl |
18 μl of 20 μM protein in 18 μl of 1x buffer (+DTT) | 36 μl | 10 μM | 16 μl | 20 μl |
16 μl of 10 μM protein in 16 μl of 1x buffer (+DTT) | 32 μl | 5 μM | 12 μl | 20 μl |
12 μl of 5 μM protein in 18 μl of 1x buffer (+DTT) | 30 μl | 2 μM | 10 μl | 20 μl |
10 μl of 2 μM protein in 10 μl of 1x buffer (+DTT) | 20 μl | 1 μM | 0 μl | 20 μl |
Made up a stock of 10x buffer with DTT: 16.3 μl 10x buffer and 1.63 μl 1 M DTT - (DTT is 10x as concentrated as before). Diluted in water as follows:
Dilution for which buffer is intended | Dilution Final Volume | Volume Buffer + DTT Stock added | Volume water added | Final volume of Buffer+water |
20 μM | 38 μl | 4.18 μl | 25.375 | 29.55 |
10 μM | 36 μl | 3.96 μl | 14.04 μl | 18 μl |
5 μM | 32 μl | 3.52 μl | 12.48 μl | 16 μl |
2 μM | 30 μl | 3.3 μl | 14.7 μl | 18 μl |
1 μM | 20 μl | 2.2 μl | 7.8 μl | 10 μl |
I put the dilutions on ice and then prepared the FA cuvettes with the following: Add 900 μl water, 100 μl 10x buffer, 1 μl 0.5 mg/ml polydIdC, and 10 μl 1 M DTT (again note that this DTT concentration is 10x last time). To one cuvette, I added 1 μl of 10 μM HmgA probe. To the second (everything else being the same), I added the same amount of Gpx1 probe. Used HmgR (purified 7.14.08) to titrate.