4 Notes
1. The components of the DSF buffer will depend on the target of
interest and its requirements. A typical DSF buffer might consist of 50 mM HEPES-Na (pH 7.5) and 100 mM NaCl. It is
advisable to avoid buffers whose pK a changes significantly with
temperature (e.g., Tris) to minimize the change in pH during
the temperature ramp. When using SYPRO
® Orange
(or similar dyes) for DSF, it is imperative that all buffers are
free of detergents as they will significantly increase the background fluorescence.
2. The fluorescence intensity of SYPRO
® Orange increases with
decreasing dielectric constant, and such is the case when it
binds to the exposed hydrophobic cores of proteins upon
thermal unfolding. Unfortunately, as a consequence of this,
SYPRO
® Orange is not suitable for protein formulations containing detergent (e.g., membrane proteins). In these situations, potential alternative dyes are CPM (N[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide
[10]) or DCVJ (9-(2,2-dicyanovinyl)julolidine [11]), which
work by different mechanisms. CPM reacts with cysteine residues within the protein of interest as it unfolds, yielding an
increase in fluorescence. DCVJ is a fluorescent molecular rotor
whose fluorescence is sensitive to viscosity and consequently
aggregation as the protein unfolds. If these dyes are investigated, the same optimization protocol described herein should
be employed.
3. An AlphaScreen antibody pair can be constructed using antibodies from any two different species, provided suitable antispecies conjugated AlphaScreen donor and acceptor beads are
available (PerkinElmer). In practice, the majority of antibodies
directed to most target proteins are raised in mouse or rabbit,
and the most efficient route to identify suitable antibody pairs is
to screen mouse-derived and rabbit-derived antibodies. The
protocol is described in terms of screening mouse- and
rabbit-derived antibodies but can be adjusted for other species
if required. The number of antibodies that are screened will
depend on the number available and any degree of prior knowledge of the target protein that may allow antibodies to be
rationally selected.
4. To validate the thermal shift of a cellular protein upon target
engagement, a well-validated tool compound is required. This
should ideally be a potent binder of the target protein, with
suitable physicochemical properties for cellular permeability
and strong evidence to support binding to the target in cells.
The compound should be tested at a concentration that
212
Joseph Shaw and Christopher Stubbs
1. The components of the DSF buffer will depend on the target of
interest and its requirements. A typical DSF buffer might consist of 50 mM HEPES-Na (pH 7.5) and 100 mM NaCl. It is
advisable to avoid buffers whose pK a changes significantly with
temperature (e.g., Tris) to minimize the change in pH during
the temperature ramp. When using SYPRO
® Orange
(or similar dyes) for DSF, it is imperative that all buffers are
free of detergents as they will significantly increase the background fluorescence.
2. The fluorescence intensity of SYPRO
® Orange increases with
decreasing dielectric constant, and such is the case when it
binds to the exposed hydrophobic cores of proteins upon
thermal unfolding. Unfortunately, as a consequence of this,
SYPRO
® Orange is not suitable for protein formulations containing detergent (e.g., membrane proteins). In these situations, potential alternative dyes are CPM (N[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide
[10]) or DCVJ (9-(2,2-dicyanovinyl)julolidine [11]), which
work by different mechanisms. CPM reacts with cysteine residues within the protein of interest as it unfolds, yielding an
increase in fluorescence. DCVJ is a fluorescent molecular rotor
whose fluorescence is sensitive to viscosity and consequently
aggregation as the protein unfolds. If these dyes are investigated, the same optimization protocol described herein should
be employed.
3. An AlphaScreen antibody pair can be constructed using antibodies from any two different species, provided suitable antispecies conjugated AlphaScreen donor and acceptor beads are
available (PerkinElmer). In practice, the majority of antibodies
directed to most target proteins are raised in mouse or rabbit,
and the most efficient route to identify suitable antibody pairs is
to screen mouse-derived and rabbit-derived antibodies. The
protocol is described in terms of screening mouse- and
rabbit-derived antibodies but can be adjusted for other species
if required. The number of antibodies that are screened will
depend on the number available and any degree of prior knowledge of the target protein that may allow antibodies to be
rationally selected.
4. To validate the thermal shift of a cellular protein upon target
engagement, a well-validated tool compound is required. This
should ideally be a potent binder of the target protein, with
suitable physicochemical properties for cellular permeability
and strong evidence to support binding to the target in cells.
The compound should be tested at a concentration that
212
Joseph Shaw and Christopher Stubbs