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|Contributor: || The Laboratory of Donald Rio at the University of California, Berkeley |
| A. Preparation of Glutathione-Agarose Beads (see Hint #2) |
1. Prepare cellular extracts that contain the Glutathione-S-Transfers (GST) fusion protein (see protocol on Preparation of Cellular Extracts) in NETN with 1 M NaCl Buffer.
2. Following the manufacture's instructions, prepare the Glutathione-Agarose beads and when completed with the manufacture's instructions, wash the beads extensively in NETN with 1 M NaCl Buffer.
B. Protein Binding to Glutathione-Agarose Beads
1. Incubate an appropriate amount of Cellular Extract with the Glutathione-Agarose beads for 45 min to 1 hour with gentle rotation at 4°C.
2. Pour the Beads containing Cellular Extract slurry into a column of appropriate size for the bed volume of beads.
3. Allow the beads to settle in the column (see Hint #3).
4. Open the flow at the bottom of the column and allow eluate to flow to waste.
5. Wash the column with 30 column volumes of NETN with 1 M NaCl Buffer.
6. Wash the column with 40 column volumes of NETN with 100 mM NaCl Buffer.
7. Wash the column with 40 column volumes of the buffer in which the pull-down experiment will be performed (e.g. Reaction Buffer, see hint Hint #4)
8. After washing extensively, place the column of Glutathione-Agarose beads in the Reaction Buffer containing 100 μg/ml Bovine Serum Albumin on ice (or at 4°C, see Hint #5).
C. Pull-Down Experiment (see Hint #4)
1. Assemble the following components in a microcentrifuge tube on ice:
150 μl of 2X Reaction Buffer.
50 μl of Kc Nuclear Extract
20 μl of a 1:1 slurry of prepared fusion protein/Glutathione-agarose beads (prepared in Section B)
80 μl ddH2O
(see Hint #6)
2. Incubate the reaction at room temperature for one hour with rotation of the microcentrifuge tubes.
3. After incubation, all tubes and solutions should be kept on ice.