Rescue of Misfolded Proteins and Stabilization by Small Molecules - Protein Misfolding and Cellular Stress in Disease and Aging - page 303

6. Unlabeled L-Met (6 mM), ribonuclease A (1.4 mg/ml), and
DNase (1.4 mg/ml; final concentrations) from Sigma to stop
the RTS assays.

1. SDS sample buffer (4×): 40% glycerol, 240 mM Tris-HCl,
pH 6.8, 8% SDS, 8% b-mercaptoethanol, and 0.04% bro-
mophenol blue.
2. Separating SDS buffer, stacking SDS buffer, 30% acrylam-
ide/0.8% bisacrylamide solution (acrylamide is a neurotoxin
when unpolymerized and so care should be taken not to
receive exposure), N , N , N , N ′-tetramethyl-ethylenediamine
(TEMED), and ammonium persulfate.
3. Sodium Dodecyl Sulphate-Polyacrylamide Gel Electrophoresis
(SDS-PAGE) equipment.
4. Autoradiography film.
5. Phosphorimager (e.g., Personal Molecular Imager from
Bio-Rad).

2.6. Sodium Dodecyl
Sulphate-
Polyacrylamide Gel
Electrophoresis
and Autoradiography

3. Methods

3.1. HTP Screening
of Stabilizing Ligands
for Soluble Proteins
by the Fluorescence
Thermal Shift Assay
(=Differential
Scanning Fluorimetry)

1. The HTP screening, searching for stabilizing ligands, is per-
formed by monitoring the thermal denaturation of proteins
in the presence of the extrinsic fluorescent probe (e.g., ANS
using a FluoDia T70).
2. The compounds from the diversity collection (e.g. from the
Maybridge HitFinder Collection database) are manually dis-
pensed into 96-well microplates with multichannel pipettes
(100 ml of 100 mM compound in 2.5% DMSO), and over-
laid with mineral oil. Robotic instrumentation can also be
used to dispense the compounds. Protein solutions, e.g.,
recombinant human PAH (0.1 mg/ml, 2 mM PAH subunit)
in 20 mM Hepes-NaOH, pH 7.0, 200 mM NaCl, and
100 mM ANS are then added. The final sample volume of
ligand, protein, and ANS in each well is 200 ml.
3. The microplates are then incubated at 25°C for 30 min before
loading onto the microplate reader.
4. Control samples without addition of DMSO and/or ligands
are routinely included in each microplate.
5. Thermal denaturation is monitored by following the increase
in ANS fluorescence intensity associated with protein
unfolding (λ exc = 395 and λ em = 500 nm, where λ exc and λ em are
the excitation and emission wavelengths, respectively)
(Figs. 2a and 2b ).