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Detection of Protein–Protein and Protein–DNA Interactions 195
protein. Second, the rapid binding, high binding capacity and low nonspecific
binding characteristics of HaloLink resin yield highly reproducible and
reliable reagent with low background signal. Furthermore, rapid binding
enables efficient immobilization of proteins at very low concentration without
the need for long incubation times. Third, HaloTag binds directly onto
HaloLink resin that eliminates the need for antibodies to precipitate
protein complexes. This is especially important for isolation of protein–DNA
complexes. Traditionally, protein–DNA complexes are isolated employing the
chromatin immunoprecipitation method (10,11). This method requires use of
specific antibodies which is the major obstacle due to lack of specific antibodies
which efficiently recognize crosslinked protein–DNA complexes. With the
HaloTag technology, formaldehyde crosslinked HaloTag protein–DNA
complexes can be isolated directly from cells using HaloLink resin, therefore
eliminating the need to use an antibody. In addition, the covalent nature of
HaloTag binding to the HaloLink resin allows very stringent washing and
removal of nonspecifically bound DNA and proteins, resulting in an increased
signal-to-noise ratio, allowing for detection of small changes in protein–DNA
interactions within the genome.
2. Materials
2.1. General Protocol for Immobilization of HaloTag Fusion
Proteins onto the HaloLink Resin
1. HaloLink resin (cat. no. G1911 or G1912, Promega).
2. TnT® quick coupled transcription/translation system (cat. no. L1170, Promega).
3. Binding buffer: 100 mM Tris–HCl pH 7.6, 150 mM NaCl, 0.05% IGEPAL-CA630
(Sigma). Warning: Solutions containing IGEPAL-CA630 should be prepared fresh
(see Note 1).
4. Wash buffer: 100 mM Tris–HCl pH 7.6, 150 mM NaCl, 1 mg/ml bovine serum
albumin (BSA), 0.05% Igepal-CA630 (Sigma) (see Notes 1 and 2).
2.2. Detection of Protein–Protein Interactions by Pre-Binding
of HaloTag Fusion Protein (Bait) to HaloLink Resin
1. HaloLink Resin (cat. no. G1911 or G1912, Promega).
2. TnT® quick coupled transcription/translation system (cat. no. L1170, Promega).
[ 35 S] methionine 2 μl (1000 Ci/mmol at 10 mCi/ml) or FluoroTect Green in vitro
Translation Labeling System (cat. no. L5001, Promega).
3. Binding buffer: Same as Subheading 2.1., step 3.
4. Wash buffer: Same as Subheading 2.1., step 4.
5. Elution buffer (4×): 0.24 M Tris–HCl (pH 6.8), 3 mM bromophenol blue, 50.4%
glycerol, 0.4 M dithiothreitol, 8% sodium dodecyl sulfate (SDS).