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198 Urh et al.
2. Carefully remove and discard the supernatant without disturbing the resin at the
bottom of the tube.
3. Add 400 μl of binding buffer, mix thoroughly by inverting the tube.
4. Centrifuge for 2 min at 800 ×gatroom temperature.
5. Carefully remove and discard the supernatant without disturbing the resin at the
bottom of the tube.
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).
3.1.3. Phase 3: Binding the HaloTag Fusion Protein
1. To the equilibrated resin, add 20 μl (or more if protein expression is low) of the in
vitro Transcription/Translation reaction containing the HaloTag fusion protein
(see Note 5).
2. Incubate by mixing on a tube rotator (see Note 6) for 30–60 min at room temperature
(incubate at 4°C if proteins are unstable, longer incubation time may be
required). 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. Save supernatant for analysis if desired.
3.1.4. Phase 4: Washing
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 for 5 min with occasional mixing.
6. Centrifuge for 2 min at 800 × g. Discard the wash.
7. Repeat steps 4–6 one more time.
8. Resuspend resin carrying covalently attached HaloTag fusion protein in desired
volume of buffer compatible with downstream applications, for example, detection
of protein interactions (see Subheadings 3.2.1, 3.2.2, 3.3. and 3.4.), analysis of
enzymatic activity (see Subheading 3.5.) of the fusion protein or cleavage of the
fusion protein from the resin (see Subheading 3.6.).
3.2. Detection of Protein–Protein Interactions In Vitro Using
Pull-Down Assay
There are two general approaches to study protein–protein interactions in
vitro using “pull-down” method. In the first approach (described in Subheading
3.2.1.), a mixture of proteins containing the HaloTag fusion proteins (from
here on referred to as bait) is added to the resin, and the bait is allowed