While we will present examples of nucleotide binding in this
protocol, other ligands can be measured by adjusting the protocol
accordingly, including larger ligands such as proteins or RNAs that
may interact with the protein of interest. It is to be noted that
additional factors may make data analysis difficult or impossible to
deconvolute and as such proper controls are required to ensure the
signal change observed reports on the appropriate reaction. In the
following protocols, the tryptophan and tyrosine residues of HflX
are excited at 280 nm and serve as donor dye(s) that can transfer
energy to the Mant group located on the nucleotide (acceptor dye)
via FRET when bound to the protein.
2 Materials
All buffers prepared in house were made using ultrapure water
(Millipore) and filtered through 0.45 μm pore size Whatman™
mixed cellulose ester membrane filters (GE Healthcare Life
Sciences).
2.1 General
1. HflX (or other protein of interest): purified as described in
Shields et al. [5].
2. Guanosine triphosphate (GTP; or other ligand of interest):
30 mM stock (Sigma Aldrich Cat. # G8877; bought as a solid).
3. Mant-GTP (or other ligand of interest): 10 mM stock (Jena
Biosciences).
2.2 Equilibrium
Nucleotide Binding
1. Guanosine triphosphate (GTP; nonfluorescently labeled nucleotide): 30 mM stock. (Sigma Aldrich Cat. # G8877; bought as
a solid).
2. Reaction buffer: 50 mM Tris–HCl pH 7.5 4
C; 70 mM KCl;
30 mM NH 4 Cl; 7 mM MgCl 2 ; filtered and degassed prior to
use (see Note 1).
3. Cuvette (Starna Cells Rectangular Quartz Fluorometer Micro
Cell with 3 mm Open Top; Cat. No: 3-3.45-Q-3).
4. Cuvette adaptor (StrataCells FCA3).
5. Long gel loading pipet tips—helpful for efficient mixing of
solution after each ligand addition.
6. Ultrapure water—filtered and degassed prior to use.
7. Acetone (Certified ACS; 99% purity).
2.3 Pre-steady State
Nucleotide Binding
1. Mant-GTP (Fluorescently labeled ligand).
2. GTP (nonfluorescently labeled ligand required for dissociation
chase experiments described in [5, 6]).
Fluorescence-Based Equilibrium and Pre-Steady State Methods
275
protocol, other ligands can be measured by adjusting the protocol
accordingly, including larger ligands such as proteins or RNAs that
may interact with the protein of interest. It is to be noted that
additional factors may make data analysis difficult or impossible to
deconvolute and as such proper controls are required to ensure the
signal change observed reports on the appropriate reaction. In the
following protocols, the tryptophan and tyrosine residues of HflX
are excited at 280 nm and serve as donor dye(s) that can transfer
energy to the Mant group located on the nucleotide (acceptor dye)
via FRET when bound to the protein.
2 Materials
All buffers prepared in house were made using ultrapure water
(Millipore) and filtered through 0.45 μm pore size Whatman™
mixed cellulose ester membrane filters (GE Healthcare Life
Sciences).
2.1 General
1. HflX (or other protein of interest): purified as described in
Shields et al. [5].
2. Guanosine triphosphate (GTP; or other ligand of interest):
30 mM stock (Sigma Aldrich Cat. # G8877; bought as a solid).
3. Mant-GTP (or other ligand of interest): 10 mM stock (Jena
Biosciences).
2.2 Equilibrium
Nucleotide Binding
1. Guanosine triphosphate (GTP; nonfluorescently labeled nucleotide): 30 mM stock. (Sigma Aldrich Cat. # G8877; bought as
a solid).
2. Reaction buffer: 50 mM Tris–HCl pH 7.5 4
C; 70 mM KCl;
30 mM NH 4 Cl; 7 mM MgCl 2 ; filtered and degassed prior to
use (see Note 1).
3. Cuvette (Starna Cells Rectangular Quartz Fluorometer Micro
Cell with 3 mm Open Top; Cat. No: 3-3.45-Q-3).
4. Cuvette adaptor (StrataCells FCA3).
5. Long gel loading pipet tips—helpful for efficient mixing of
solution after each ligand addition.
6. Ultrapure water—filtered and degassed prior to use.
7. Acetone (Certified ACS; 99% purity).
2.3 Pre-steady State
Nucleotide Binding
1. Mant-GTP (Fluorescently labeled ligand).
2. GTP (nonfluorescently labeled ligand required for dissociation
chase experiments described in [5, 6]).
Fluorescence-Based Equilibrium and Pre-Steady State Methods
275