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

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 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 ~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 μM	38 μl	4.18 μl	20.24	24.42
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

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.

Data

August 8, 2008 GpxR Data