DNA pull-down assays

RATIONALE I have recently purified ScCdt1 and now have it in my regular Pre-RC sizing buffer (50 mM HEPES pH 7.5, 300 mM KGlutamate, 10% glycerol, 1 mM BME). Now that I have this, I want to repeat the DNA pull down I did over a year ago now comparing ScCdt1 and DmCdt1 DNA binding. I’m repeating it since last time I did it in a different buffer and for this paper I want to have every single one of my proteins in the same buffer. I’ll repeat the assay nearly exactly as I recently completed it for Cdc6 (103117_DNApulldownDmCdc6), using the same beads that were prepared for this previous experiment.

EXPERIMENTAL DESIGN I have the following protein and buffer stocks:

Assay Buffer: 50 mM HEPES pH 7.5, 150 mM KGlutamate, 10% glycerol, 1 mM BME

Dilution Buffer: 50 mM HEPES pH 7.5, 10% glycerol, 1 mM BME

Protein Buffer: 50 mM HEPES pH 7.5, 10% glycerol, 300 mM KGlutamate, 1 mM BME

ScCdt1: 32 uM in Protein Buffer

DmCdt1: 100 uM in Protein Buffer

I prepared the following stocks which, after preparation, are in Assay Buffer (150 mM KGlut):

140 uL 10 uM ScCdt1: 43.75 uL ScCdt1 + 26.25 uL Protein Buffer + 70 uL Dilution Buffer

140 uL 10 uM DmCdt1: 14 uL DmCdt1 + 56 uL Protein Buffer + 70 uL Dilution Buffer

1. Fully resuspend the prepared control and DNA-coupled agarose beads
2. Transfer 60 uL (30 uL of beads) of control beads to two tubes and 60 uL of DNA beads to two tubes
3. Centrifuge 2500 xg 2 min and remove supernatant
4. Wash the beads with 100 uL of Assay Buffer, centrifuge, and remove supernatant
5. Repeat Step C
6. Prepare samples as follows:
   1. Control beads + 50 uL 10 uM ScCdt1
   2. Control beads + 50 uL 10 uM DmCdt1
   3. DNA beads + 50 uL 10 uM ScCdt1
   4. DNA beads + 50 uL 10 uM DmCdt1
7. Incubate for 30 minutes at room temperature with occasional agitation
8. Centrifuge 2500 xg 2 min and remove (and save) supernatant
9. Wash with 500 uL of Assay Buffer, centrifuge, and remove supernatant
10. Repeat Step I two more times (for a total of three washes)
11. Resuspend the beads with 50 uL of 1x reducing SDS-PAGE loading buffer
12. Boil the sample for 3 minutes, centrifuge at max speed for 30 sec, and then transfer the supernatant to a fresh tube for SDS-PAGE analysis

Using the remaining 10 uM stock I prepared an SDS-PAGE sample using 5x reducing buffer to run as a “Load” sample. I ran 4 and 2 uL of this “Load” sample, and 8, 4, and 2 uL of the Samples from L on a 4-20% SDS-PAGE gel.