HeLa cells (1 × 10 cells/mL buffer solution) were incubated with carbonyl cyanine m-chlorophenylhydrazone (CCCP; 0, 0.25 0.55, 0.75 0.75, 1, 2.5, 5, 10, and 25 μmol/L) for 5 min at 37°C in 1.5 mL microtubes, followed by the addition of the IC7-1 derivative (1 μmol/L final concentration).
The remaining portion of the labeling reaction was added to 16 ml of the standard hybridization buffer, the resulting solution was incubated in a water bath at 95°C for 10 min.
DHCC in a 20 mM Tris-HCl buffer (pH 7.5) solution was incubated with either sodium dithionite (reductant) or potassium ferricyanide (oxidant) at a final concentration of 25 μM for both the protein and the reductant or oxidant.
The recombinant protein GP73 was diluted into 2 μg/mL in binding buffer and 200 μL of solution was incubated in a 96-well microtiter plate overnight at 4°C.
Briefly, cells were washed twice with cold PBS, resuspended in binding buffer (1×106 cells/ml), 100 µl of solution was incubated with 5 µl of Annexin V-PE and 5 µl of 7-AAD for 15 min at RT in the dark, and 400 µl of binding buffer was added and cells analyzed by flow cytometry.
First, the recombinant GP73 protein or fetal bovine serum (FBS) was diluted to a series of concentrations (31.25 ng/mL–4 μg/mL) in binding buffer and 200 μL of each solution was incubated in a 96-well microtiter plate overnight at 4°C.
S1, S2 and T were mixed in a phosphate buffer (1M NaCl, 1mM MgCl2), and the solution was incubated at 95 °C, rapidly cooled to 4 °C, and again incubated at 4 °C for 20 min. The volume of the solution was 50 μL and the concentrations were controlled in each experiment.
For the measurement of vesicle-induced changes in the emission spectra of tryptophan in Trp-containing peptides, a 2-µM peptide solution was incubated in buffer A, and aliquots of vesicles suspension were added to give total lipid concentrations in the range 0 to 300 µM total lipid.
Synbodies were screened on the array using the recommended protocol with the following modifications: the arrays were blocked with 5 mL blocking buffer (50mM HEPES pH 7.5, 200 mM NaCl, 0.08% Triton X-100, 25% Glycerol, 20mM reduced Glutathione, 1.0mM DTT, 1% BSA) for 90 min. A 200 nM synbody solution was incubated in probing buffer (1× PBS, 1% BSA, 0.1% Tween) for 90 minutes.
For the spectra of a refolded protein, the protein that was completely unfolded at a 7.0 M GdnHCl concentration was diluted with buffer for refolding, and the diluted protein solution was incubated at the selected temperature until refolding reached equilibrium.
Coverslips were washed and incubated in 1X PBS pH 7.4 containing 5% FBS and 3% BSA (blocking solution) for at least 30 min. Primary antibody diluted in blocking solution was incubated for 30 60 min, the coverslips washed 3X with blocking buffer and then incubated for 30 60 min with secondary antibodies: Alexa Fluor 594 for mouse diluted 1 2000 in blocking buffer.
