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 μl20 μM18 μl20 μl
18 μl of 20 μM protein in 18 μl of 1x buffer (+DTT)36 μl10 μM16 μl20 μl
16 μl of 10 μM protein in 16 μl of 1x buffer (+DTT)32 μl5 μM12 μl20 μl
12 μl of 5 μM protein in 18 μl of 1x buffer (+DTT)30 μl2 μM10 μl20 μl
10 μl of 2 μM protein in 10 μl of 1x buffer (+DTT)20 μl1 μM0 μl20 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 μM38 μl4.18 μl25.375 29.55
10 μM36 μl3.96 μl14.04 μl18 μl
5 μM32 μl3.52 μl12.48 μl16 μl
2 μM30 μl3.3 μl14.7 μl18 μl
1 μM20 μl2.2 μl7.8 μl10 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.

Data

August 8, 2008 HmgR* Data

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