Matt Gethers/CRI, Thailand/Labwork/Binding Assay
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Protein DNA Binding Assay using Fluorescence Anisotropy
- Anneal BT2767 and BT2768 to form the HmgA promter probe and anneal BT2769 and BT2770 to form the Gpx1 promoter probe. Add 10 μl of each primer to 80 μl of water, boil at 100 oC for 5 minutes, then turn off heat block and allow to cool to room temperature.
- Prepare binding reaction buffer (see below).
- Prepare dilutions of peptide. Aim for 20, 10, 5, 2, and 1 μM. Do serial dilutions. See the table for a suggested dilution pattern.
- Place dilutions on ice and get polydIdC and 1 M DTT.
- Get cuvettes from the room with the FA equipment. Add 900 μl water, 100 μl 10x buffer, 1 μl 0.5 mg/ml polydIdC, and 1 μl 1 M DTT.
- Mix the contents of the cuvette, clean with a kim wipe, and place in the reader to measure intensity and basal anisotropy.
- Begin by adding 1 μl samples of 1 μM dilution to the cuvette. Mix and place in reader. Continue by adding more of 1 μM, then move on to 2, 5, 10, and 20.
10x Binding Reaction Buffer
|Tris pH 7.0||200 mM|
|Calf Thymus DNA||50 μg/ml|
|Volumes and Concentrations of Constituents||Volume||Final Concentration||Volume removed for next dilution||Final Volume|
|x μl of stock protein in y 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|
- Note: Because you're adding different volumes of buffer to each dilution, you can't make a stock of a particular concentration and use in each. Make up a stock of 10x buffer with DTT (16.3 μl 10x buffer and 1.63 μl 100 mM DTT will make enough for the 5 dilutions outlined in the table), then add different amounts of this stock and water to each dilution. Table follows:
|Dilution||Dilution Final Volume||Volume Buffer + DTT Stock added||Volume water added||Final volume of Buffer+water|
|20 μM||38 μl||4.18 μl||varies||varies|
|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|