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Detection of Protein–Protein and Protein–DNA Interactions 205
3.3.3.3. Phase 3: Capture of Protein Complexes
1. To the resin, add 100 μl of the cytosol prepared as described above (preparation of
cytosolic fraction; the volume of the cytosolic fraction may have to be adjusted,
use 100 μl only as a guideline).
2. Incubate with mixing using rotation for 1hatroom temperature or 4hat4°C
(see Note 9). Make sure resin does not settle to the bottom of the tube as that will
reduce efficiency of binding.
3. Centrifuge for 2 min at 800 × g.
3.3.3.4. Washing
1. Add 1 ml of wash buffer containing 0.5 mg/ml BSA and mix thoroughly by
inverting the tube.
2. Centrifuge for 2 min at 800 × g. Discard the wash.
3. Repeat steps 1 and 2 two more times.
4. Add 1 ml of wash buffer containing 0.5 mg/ml BSA and mix thoroughly by
inverting the tube.
5. Incubate 5 min with occasional mixing.
6. Centrifuge for 2 min at 800 × g. Discard the wash.
7. Repeat steps 4–6 twice one more time.
3.3.3.5. Elution
1. Add 30 μl of 1× SDS loading buffer and heat to 95°C for 2–5 min.
2. Remove supernatant and analyze samples immediately or store at –20°C. Proteins
can be resolved on a SDS–PAGE gel and analyzed by Western blotting.
3.4. Detection of Protein–DNA Interactions
This protocol is designed for the use of 1–5 × 10 6 cells at 70–80% confluency.
Typically, this is 1–2 wells of a 6-well plate, each containing 2 ml of cells (see
Note 11).
3.4.1. Resin Equilibration
1. Aliquot 100 μl of HaloLink resin into a 1.5-ml microcentrifuge tube.
2. Centrifuge resin for 3 min at 800 × g and remove the supernatant.
3. Wash resin with 400 μl HaloLink Equilibration Buffer.
4. Centrifuge for 3 min at 800 × g and remove the wash.
5. Repeat steps 3 and 4 two more times.
6. Remove the final wash and add 100 μl of 1× TBS (BSA at a final concentration
of 1 mg/ml may be added if desired).
3.4.2. Crosslinking, Capture and Release of DNA
1. Grow approximately 1×10 6 cells to 70–80% confluency.
2. With constant swirling, slowly add formaldehyde (stock concentration of 37%)
to a final concentration of 1% directly to cells.