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Detection of Protein–Protein and Protein–DNA Interactions 203
6. Repeat steps 3–5 two more times for a total of three washes.
7. After last wash, resuspend the resin in 50–100 μl of binding buffer (see Note 4).
Add co-factors, detergents or other reagents needed for specific protein–protein
interactions.
3.2.2.3.2. Bait–Prey Complex Capture and Analysis
1. To the resin samples, add 20 μl of the appropriate mix (experimental bait–prey or
control) set up above (see Subheading 3.2.2.2.).
2. Incubate by mixing on a tube rotator (see Notes 6 and 9) for 1–2 h at room
temperature. 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 and discard the supernatant.
3.2.2.3.3. Washing
Stability of different protein–protein interactions is protein pair specific and
depends on the affinity of interaction. If interaction is not very stable, the
washing conditions used for these protein pairs may have to be optimized,
for example, change the number and volume of washes. However, insufficient
washing may result in detection of nonspecific interactions.
1. Add 1 ml of wash buffer containing 1 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 1 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 one more time.
3.2.2.3.4. Elution
1. Add 20 μl of 1× SDS loading buffer (for composition see Subheading 2; 0.5 or
0.25 × elution buffer can also be used).
2. Incubate 2–5 min at 90°C (see Note 10).
3. Remove supernatant and load on a SDS–PAGE gel for analysis.
3.3. Detection of Protein–Protein Interactions In Vivo
This protocol is intended to serve as a guide. You should empirically optimize
the cell culture protocol, transfection conditions, amount of HaloLink resin
used and adjust buffers if necessary.