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196 Urh et al.
2.3. Detection of Protein–Protein Interactions by Isolation
of Pre-Formed Bait–Prey Complexes
Follow the steps as in Subheading 2.2.
2.4. Detection of Protein–Protein Interactions In Vivo
1. HaloLink Resin (cat. no. G1911 or G1912, Promega).
2. Binding buffer: Same as Subheading 2.1., step 3, except the concentration of
IGEPAL-CA630 is reduced to 0.001%.
3. Wash buffer: Same as Subheading 2.1., step 4, except the concentration of BSA
is reduced to 0.5%.
4. Elution buffer: Same as Subheading 2.2., step 5.
2.5. Detection of Protein–DNA Interactions
1. HaloLink resin (cat. no. G1911 or G1912, Promega).
2. HaloLink Equilibration Buffer: 1× Tris-EDTA buffer (TE) pH 7 (10 mM Tris–
HCl pH 7.0, 1 mM EDTA) 0.05% IGEPAL or 0.5% Triton X-100.
3. Tris buffered saline (TBS) buffer, 1×: 100 mM Tris–HCl pH 7.6, 150 mM NaCl.
4. Phosphate-buffered saline (PBS) buffer, Dulbecco’s PBS, 1× (cat. no. 14190,
Invitrogen).
5. Lysis buffer: 50 mM Tris–HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, 1% Triton
X-100, 0.1% sodium deoxycholate (NaDOC).
6. High salt lysis buffer: 50 mM Tris–HCl pH 7.5, 700 mM NaCl, 5 mM EDTA,
1%, Triton X-100, 0.1% NaDOC.
7. Reversal buffer: 1× TE pH 7, 300 mM NaCl.
2.6. Enzyme Immobilization and Analysis of Enzymatic Activity
on the Surface
1. HaloLink Resin (cat. no. G1911 or G1912, Promega).
2. Binding buffer: Same as Subheading 2.1., step 3.
3. Wash buffer: Same as Subheading 2.1., step 4.
2.7. One-Step Purification of Fusion Proteins
1. HaloLink Resin (cat. no. G1911 or G1912, Promega).
2. Binding buffer: Same as Subheading 2.1., step 3.
3. Wash buffer: Same as Subheading 2.1., step 4.
2.8. Cloning Vectors
The HaloTag-containing Flexi® Vectors are available for the cloning of
desired proteins. The protein of interest can be fused to HaloTag using Flexi®