The behavior of Ptc proteins was analyzed by CD, CPM fluorescence thermal stability assays, and by analytical SEC using a Superose6 column. For CD analysis, spectra were collected at 4°C between 185 nm and 280 nm, using Ptc proteins in a cuvette with 1 mm pathlength. Thermal unfolding analysis was also performed by following the CD signal at 208 nm while increasing the temperature from 4°C to 95°C. CPM fluorescence thermal stability assays were performed as in [35 (link)]. In brief, a 1:40 dilution of a 4 mg/ml solution of CPM dye (Invitrogen) in DMSO was incubated in CPM Thermal Melt Buffer for 5 min at room temperature, protected from light. Ptc protein (10 μg) was diluted in CPM Thermal Melt Buffer to a final volume of 290 μL. After 5 min at room temperature, 10 μL of dilute dye was added and the sample was mixed and transferred to a quartz fluorometer cuvette. The cuvette was transferred to a Fluorolog-3 spectrofluorometer (Horiba Jobin Yvon) equipped with a Peltier sample cooler (F-3004) and heated at a rate of 2°C/min. Emission at 463 nm was monitored with an excitation wavelength of 387 nm from 20°C to 80°C. For SEC analysis, Ptc was injected onto a Superose6 column equilibrated in Pulldown Buffer. Its elution profile was followed by UV absorption at 280 nm, and apparent molecular weights were estimated by comparison to Gel Filtration Standards (Bio-Rad).
Cpm dye
Manufactured by Thermo Fisher Scientific
CPM dye is a fluorescent labeling reagent used in molecular biology for the detection and quantification of proteins, nucleic acids, and other biomolecules. It functions by covalently binding to target molecules, allowing their visualization and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Dimethyl sulfoxide (dmso), by Merck Group (3 mentions)
Fluorolog 3 spectrofluorometer, by Horiba (1 mentions)
Gel filtration standard, by Bio-Rad (1 mentions)
100 kda molecular weight cutoff filter, by Merck Group (1 mentions)
Cary eclipse spectrofluorometer, by Agilent Technologies (1 mentions)
Prism software, by GraphPad (1 mentions)
Lightcycler 480 system, by Roche (1 mentions)
4 protocols using cpm dye
1
Characterization of Ptc Protein Behavior
2
Purification and Stability Analysis of UapA-GFP
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UapAG411VΔ1–11 from Aspergillus nidulans was expressed as a C-terminal GFP fusion protein in the FGY217 strain of Saccharomyces cerevisiae. The UapA was isolated and purified in sample buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 1 mM xanthine) according to the reported protocol52 (link). The protein was concentrated to approximately 10 mg/mL using a 100 kDa molecular weight cut off filter (Millipore). The protein was diluted 1:150 into buffer containing either TMG-As (TMG-A11, TMG-A12, TMG-A13 and TMG-A14), TMG-Ts (TMG-T11, TMG-T12, TMG-T13 and TMG-T14) or DDM to give final detergent concentrations of CMC + 0.04 wt% or CMC + 0.2 wt% in Greiner 96-well plates. The CPM dye (Invitrogen) stored in DMSO (Sigma) was diluted in dye buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 0.03% DDM, 5 mM EDTA) and 3 μL of the dye buffer was added to each sample. Protein stability was measured by incubating the reaction mixture for 125 min at 40 °C, starting from 30 min after sample dilution. The fluorescence emission was recorded using a microplate spectrofluorometer set at excitation and emission wavelengths of 387 nm and 463 nm, respectively. The relative amounts of folded proteins were plotted against time using GraphPad Prism.
3
CPM Thermal Denaturation Assay
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The CPM stability assay was performed as follow36 (link). A 4 mg ml−1 stock solution of CPM dye (Invitrogen) was prepared in dimethylsulfoxide (Sigma) and diluted to 0.1 mg ml−1 in dilution buffer (20 mM HEPES (pH 7.5) and 150 mM NaCl) before use. The thermal denaturation assay was performed in a total volume of 130 μl. The ClR protein and its mutants (F15A, W72A, Y83A and delta C-terminal helix; 1–254) were diluted in the appropriate buffer to a final volume of 120 μl. Ten microlitre of the diluted dye was added and thoroughly mixed with the protein. The reaction mixture was transferred within a 5 min period to a sub-micro quartz fluorometer cuvette (Stana Cells, Inc., Atascadere, CA) and heated in a controlled way with a ramp rate of 2 °C min−1 in a Cary Eclipse Spectrofluorometer. The excitation wavelength was set at 387 nm, and the emission wavelength was set at 463 nm. Assays were performed over a temperature range beginning at 30 °C and ending at 90 °C. The stability data were processed using GraphPad Prism software (GraphPad Software, San Diego, CA).
4
Thermal Stability Assay of ABCG1 Variants
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The thermal stability assay was performed using a previously described technique with slight modification (Alexandrov et al., 2008) . CPM dye (Invitrogen) was dissolved in DMSO (Sigma) at 4 mg/ml. This stock solution was kept at À80 C. Prior to use, the dye stock is diluted 1:100 in dye dilution buffer (30 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.01% LMNG and 0.001% CHS), and is used immediately while protected from light to reduce photobleaching. The thermal denaturation assay was performed in a total volume of 30 mL. The purified samples of ABCG1 variants (10 mg) was diluted in the dye dilution buffer to a final volume of 24 mL. Then, 6 mL of the diluted dye was added and thoroughly mixed with the protein samples. The reaction mixture was heated in a controlled manner with a ramp rate of 6 C/min in a LightCyclerâ 480 System (Roche). The excitation wavelength was set at 440 nm, and the emission wavelength was 488 nm. Assays were performed over a temperature range starting from 37 C and ending at 80 C.
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