Matt Gethers/CRI, Thailand/Labwork/Binding Assay/8.7.08 Run Notes

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8.7.08 Binding Assay Run

Ran experiment testing GpxR binding affinity for both the HmgA and Gpx1 promoter regions. Got 10x binding buffer from p'So and made dilutions of lot 1 of GpxR as follows:

Volumes and Concentrations of Constituents Volume Final Concentration Volume removed for next dilution Final Volume
13.57 μl of stock protein (assume 56 μM dimer) in 24.42 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 ~18 μl of 10x Buffer+DTT using 16.3 μl 10x and 1.63 μl 100 mM DTT. Diluted 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 μl20.24 24.42
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

Placed dilutions on ice, then to an FA cuvette added: 900 μl MilliQ water, 100 μl 10x buffer, 1 μl 0.5 mg/ml polydIdC, and 1 μl 1 M DTT. To first cuvette, added 1 μl 10 μM Gpx1 promoter probe (final concentration of 10 nm). Added the same amount of HmgA promoter to second cuvette which otherwise had all the same reagents in the volumes stated above.


August 8, 2008 GpxR Data

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