View - ResearchGate

208 Urh et al.
by 0.5% Triton X-100 or by 5% glycerol. BSA may also be eliminated if it
interferes with the activity of the protein, but higher nonspecific binding may be
detected. Other Tris-based buffers can be used in this protocol.
3. Appropriate speed in rpm can be calculated from the following formula, RCF =
(1.12)(r)(rpm/1000) 2 where r = radius in mm measured form the center of spindle
to bottom of rotor bucket; rpm = revolutions per minute. In a standard size
microcentrifuge, 800 × g corresponds to 3000 rpm.
4. Volume used for resuspending the resin can be adjusted for a specific experiment.
5. In case of proteins expressed in mammalian cells, we added 100 μl of cytosolic
fraction to 50 μl of the HaloLink resin.
6. We used a tube rotator from Scientific Equipment Products; other mixing devices
can be used (e.g., IKA-SCHÜTTLER MTS2).
7. In vitro Transcription/Translation (TnT®) reactions are typically 50 μl, which
may be sufficient for more than one pull-down reaction. Efficiency of the in
vitro protein synthesis and the strength of protein–protein interaction may differ
for different protein pairs, thus, the volume of the in vitro TnT® reaction added
to the HaloLink resin may have to be adjusted for a specific pair. Smaller or
larger volumes may be needed.
8. If immobilization of proteins onto HaloLink takes longer than incubation time
required for TnT® T7 Quick Coupled Transcription/Translation, it is best to
keep reactions at 30°C or on ice, if protein stability is in question. Prolonged
incubation on ice may result in protein precipitation. An aliquot of 1–5 μl of
the reaction may be saved for analysis of the efficiency of the prey synthesis by
SDS–PAGE gel.
9. Time of incubation may need optimization for different protein pairs.
10. Overheating may result in aberrant migration of proteins or even prevent protein
migration into the gel. If this occurs, heat samples to 70°C for 3–5 min or 60°C
for 10 min. When analyzing the efficiency of the prey synthesis, too much of
the sample may cause coagulation of hemoglobin and cause aberrant migration
in the gel. We suggest to reduce the volume of reaction loaded to 1–2 μl.
11. When using 0.1–0.5 × 10 6 cells, reduce the amount of HaloLink resin to 50–75
μl. When using 0.5–1 × 10 7 cells, increase the amount of HaloLink resin to
125 μl.
References
1. Zhang, J., Campbell, R. E., Ting, A. Y., and Tsien, R. Y. Creating new fluorescent
probes for cell biology. (2002) Nat. Rev. Mol. Cell Biol. 3, 906–918.
2. Lippincott-Schwartz, J. and Patterson, G. H. Development and use of fluorescent
protein markers in living cells. (2003) Science 300, 87–91.
3. Miyawaki, A., Sawano, A., and Kogure, T. Lighting up cells: labelling proteins
with fluorophores. (2003) Nat. Cell Biol. 5, Suppl., S1–S7.
4. Porath, J., Carlsson, J., Olsson, I., and Belfrage, G. Metal chelate affinity
chromatography, a new approach to protein fractionation. (1975) Nature 258,
598–599.