by partial unfolding and oligomerization) to a more desirable single
transition.
2.3.3 Thermofluor/DSF
of Extrinsic Fluorophores
Various fluorescent dyes are available to probe changes in protein
conformation [38], and any of these may be used as long as the
optical system of the instrument is compatible with their absorption
and fluorescent emission properties. Common dyes are Nile red
with a maximum excitation at 550–580 nm and emission in the
range 590–690 nm (environment dependent), 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) with 370 nm excitation and
450–550 nm emission, and 4,4
0 -dianilino-1,1
0 -binaphthyl-5,50 -disulfonic acid (bis-ANS) with 385 nm excitation/450–550 nm
emission. SYPRO Orange is the most widely used dye for working
with soluble proteins [45] because it has excitation and emission
(460–520/550–650 nm) wavelengths that are compatible with
almost all qPCR machines.
Thermofluor can also be applied to membrane proteins. However, interactions of the dye with the apolar external surface of the
membrane protein or with detergents/amphipols may lead to a
high initial signal background making changes in signal difficult
to measure. Alternative dyes with lower background signal have
been developed such as the thiol-specific fluorochrome N-[4
(7-diethylamino)-4-methyl-3-coumarinyl] maleimide (CPM)
(385/470 nm) and Proteostat
® dye (530/560–650 nm)
[28]. Only a subset of qPCR instruments have optics suitable for
use with these dyes.
In a thermofluor assay, the total volume per well is 25μL. Each
condition tested requires 2μL of purified protein at an initial concentration of 20μM. SYPRO Orange (Invitrogen) is delivered as a
5000Â concentrated stock solution in DMSO and needs to be
diluted to a 62.5Â solution in ultrapure water (3μL of SYPRO
5000Â and 237μL of water), and 2μL of 62.5Â SYPRO Orange
is added per well. Do not premix the protein and the dye (the high
concentration of DMSO may damage the protein) and follow the
order of addition of components below for best results.
A simple protocol for buffer optimization via thermofluor is
given below:
1. Put the plate on ice to equilibrate the sample at the starting
temperature of the experiment.
2. Put sufficient buffer into each well to have finally a total volume
of 25μL.
3. Add 2μL of 10Â additive if needed.
4. Add 2μL of 20μM protein.
5. Add 2μL of 62.5Â SYPRO Orange solution.
6. Seal the plate with highly transparent optical-clear quality
sealing tape.
Protein Quality Control
37
transition.
2.3.3 Thermofluor/DSF
of Extrinsic Fluorophores
Various fluorescent dyes are available to probe changes in protein
conformation [38], and any of these may be used as long as the
optical system of the instrument is compatible with their absorption
and fluorescent emission properties. Common dyes are Nile red
with a maximum excitation at 550–580 nm and emission in the
range 590–690 nm (environment dependent), 1-anilinonaphthalene-8-sulfonic acid (1,8-ANS) with 370 nm excitation and
450–550 nm emission, and 4,4
0 -dianilino-1,1
0 -binaphthyl-5,50 -disulfonic acid (bis-ANS) with 385 nm excitation/450–550 nm
emission. SYPRO Orange is the most widely used dye for working
with soluble proteins [45] because it has excitation and emission
(460–520/550–650 nm) wavelengths that are compatible with
almost all qPCR machines.
Thermofluor can also be applied to membrane proteins. However, interactions of the dye with the apolar external surface of the
membrane protein or with detergents/amphipols may lead to a
high initial signal background making changes in signal difficult
to measure. Alternative dyes with lower background signal have
been developed such as the thiol-specific fluorochrome N-[4
(7-diethylamino)-4-methyl-3-coumarinyl] maleimide (CPM)
(385/470 nm) and Proteostat
® dye (530/560–650 nm)
[28]. Only a subset of qPCR instruments have optics suitable for
use with these dyes.
In a thermofluor assay, the total volume per well is 25μL. Each
condition tested requires 2μL of purified protein at an initial concentration of 20μM. SYPRO Orange (Invitrogen) is delivered as a
5000Â concentrated stock solution in DMSO and needs to be
diluted to a 62.5Â solution in ultrapure water (3μL of SYPRO
5000Â and 237μL of water), and 2μL of 62.5Â SYPRO Orange
is added per well. Do not premix the protein and the dye (the high
concentration of DMSO may damage the protein) and follow the
order of addition of components below for best results.
A simple protocol for buffer optimization via thermofluor is
given below:
1. Put the plate on ice to equilibrate the sample at the starting
temperature of the experiment.
2. Put sufficient buffer into each well to have finally a total volume
of 25μL.
3. Add 2μL of 10Â additive if needed.
4. Add 2μL of 20μM protein.
5. Add 2μL of 62.5Â SYPRO Orange solution.
6. Seal the plate with highly transparent optical-clear quality
sealing tape.
Protein Quality Control
37