Absorbance measurements were performed in 1 mL quartz cuvettes. HaloTag protein was used as a 100 μM solution in 75 mM NaCl, 50 mM TRIS·HCl, pH 7.4 with 50% v/v glycerol (TBS–glycerol). HaloTag ligands 27 and 28 (5 μM) were dissolved in 10 mM HEPES, pH 7.3 containing 0.1 mg·mL−1 CHAPS. An aliquot of HaloTag protein (1.5 equiv) or an equivalent volume of TBS–glycerol blank was added and the resulting mixture was incubated until consistent absorbance signal was observed (~30 min). Additional HaloTag protein did not elicit an increase in absorbance (not shown). Absorbance scans are averages (n = 2).
Protocol detail
HaloTag Ligand Absorbance Measurement
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Corresponding Organization :
Other organizations : Janelia Research Campus, Howard Hughes Medical Institute, Physique des Cellules et Cancers, Institut Curie
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Variable analysis
independent variables
- HaloTag ligands 27 and 28 (5 μM)
- Absorbance measurements
- HaloTag protein (100 μM solution in 75 mM NaCl, 50 mM TRIS·HCl, pH 7.4 with 50% v/v glycerol (TBS–glycerol))
- HaloTag ligands dissolved in 10 mM HEPES, pH 7.3 containing 0.1 mg·mL^-1 CHAPS
- Incubation time (~30 min)
- HaloTag protein (1.5 equiv)
- TBS–glycerol blank
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