The thermal stability of purified protein was determined by measuring fluorescence of thiol-reactive dye BODIPY FL-L-cystine (Invitrogen). The standard assay conditions were 20 mM HEPES (pH 7.5), 200 mM NaCl, 0.025% DDM and 10 mM risperidone with protein concentrations 1 mg/mL and BODIPY FL-L-cystine concentrations 1 μM. The melting experiments were performed on a StepOnePlus real-time PCR System from Applied Biosystems. The melting curve experiments were conducted (1 °C/min) and recorded using StepOne software from Applied Biosystems. Results were analyzed using GraphPad Prism. Apparent Tm values were derived from sigmoidal dose–response analysis. Results represent the mean ± SEM of three independent experiments.
Bodipy fl l cystine
Manufactured by Thermo Fisher Scientific
Sourced in Italy
BODIPY FL 1-cystine is a fluorescent dye used for labeling and detecting cysteine-containing proteins. It has an excitation maximum at 503 nm and an emission maximum at 512 nm. The dye can be used for various applications, such as protein analysis and cellular imaging.
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Lab products found in correlation
Stepone software, by Thermo Fisher Scientific (4 mentions)
Steponeplus real time pcr system, by Thermo Fisher Scientific (4 mentions)
Sypro orange, by Thermo Fisher Scientific (2 mentions)
Synergy mx plate reader, by Agilent Technologies (1 mentions)
Ethanol, by Merck Group (1 mentions)
Ethylenediaminetetraacetic acid, by Merck Group (1 mentions)
1 2 distearoyl sn glycero 3 phosphocholine, by Avanti Polar Lipids (1 mentions)
Aldolase, by Merck Group (1 mentions)
Dithiothreitol (dtt), by GoldBio (1 mentions)
Pyruvate kinase, by Merck Group (1 mentions)
3 4 methylenedioxy β nitrostyrene, by Merck Group (1 mentions)
N ethylmaleimide, by Merck Group (1 mentions)
Glutathione (gsh), by Merck Group (1 mentions)
Fluostar optima microplate reader, by BMG Labtech (1 mentions)
Bovine carbonic anhydrase 2, by Merck Group (1 mentions)
9 protocols using bodipy fl l cystine
1
Thermal Stability Analysis of Purified Protein
2
Thermal Stability Analysis of Purified Protein
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The thermal stability of purified protein was determined by measuring fluorescence of thiol-reactive dye BODIPY FL-L-cystine (Invitrogen). The standard assay conditions were 20 mM HEPES (pH 7.5), 200 mM NaCl, 0.025% DDM and 10 mM risperidone with protein concentrations 1 mg/mL and BODIPY FL-L-cystine concentrations 1 μM. The melting experiments were performed on a StepOnePlus real-time PCR System from Applied Biosystems. The melting curve experiments were conducted (1 °C/min) and recorded using StepOne software from Applied Biosystems. Results were analyzed using GraphPad Prism. Apparent Tm values were derived from sigmoidal dose–response analysis. Results represent the mean ± SEM of three independent experiments.
3
Quantifying Thiol-Drug Cystine Cleavage
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BODIPY FL L-cystine (Thermo Fischer scientific) was reconstituted in methanol. In a black Maxi Sorp 96-well flat-bottomed plate (Nunc), 10 μM of BODIPY reagent prepared in PBS was mixed with 25 μM of the thiol-based drugs and the change in fluorescence was kinetically measured with excitation at 490nm and emission at 520nm, at 1minute intervals, for one hour at 37°C. Fluorescence reads, after subtracting the drug-free control reads, were plotted against time. The maximum slope (Max V) over a 10 minute interval for all the thiol-based drugs from this plot was calculated and represented as relative fluorescence units/min (RFU/min) to assess the cystine cleaving ability of the drugs. The experiment was repeated three times.
4
Recombinant GILT Disulfide Reduction Assay
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Recombinant mosGILT activity was assessed by the ability of the enzyme to reduce the disulfide bond of a fluorescent molecule, BODIPY FL L-Cystine (Thermo Fisher Scientific) as previously described63 (link). Purified rGILT, human GILT, mouse GILT C2 (CxxS), and lysozyme at 0.05 μM were preactivated with DTT (25 μM) for 10 min at room temperature in a sodium-acetate buffer pH 4.5 or PBS pH 7.4. Serially diluted BODIPY FL L-Cystine substrate (30 μM to 0.234 μM) was added to the enzyme mixture in a 96-well plate (Microfluor 1) and relative fluorescence was monitored over time at 28 °C using a Synergy Mx plate reader (BioTek) with emission/excitation settings of 330/580.
5
Nanoparticle Lipid Formulation Protocol
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1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), 1,2-dipalmitoyl-sn-glycero-3-phospho-(1′-rac-glycerol) sodium salt
(DPPG-Na), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt (DSPE-PEG2000-Mal)
were purchased from Avanti Polar Lipids, Alabaster, Alabama (USA). tert-Butanol (t-BuOH), palmitic acid (PA),
poly(ethylene glycol) with molecular weight 3500–4500 (PEG
4000), ethylenediaminetetraacetic acid (EDTA), phosphate-buffered
saline (PBS, pH 7.4), and ethanol were Sigma-Aldrich, Milan, (Italy)
products. BODIPY FLl -cystine and gel-immobilized (tris(2-carboxyethyl)phosphine)
disulfide (TCEP) reducing agent were purchased from Thermo Fisher
Scientific, Monza, MB (Italy). cyclo-(Arg-Gly-Asp-D-Phe-Cys)
(c-RGDfC) purchased from Peptides International,
Louisville, Kentucky (USA), sulfur hexafluoride (SF6) from
Rivoira Gas, Rome, RM (Italy), and ICG from Pulsion Medical System,
Munich (Germany) were used without further purification. Water of
Milli-Q purity grade (18.2 MΩ·cm) was produced with an
Elga PureLab Classic, High Wycombe, (UK) deionization apparatus.
(DPPG-Na), and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[maleimide(polyethylene glycol)-2000] ammonium salt (DSPE-PEG2000-Mal)
were purchased from Avanti Polar Lipids, Alabaster, Alabama (USA). tert-Butanol (t-BuOH), palmitic acid (PA),
poly(ethylene glycol) with molecular weight 3500–4500 (PEG
4000), ethylenediaminetetraacetic acid (EDTA), phosphate-buffered
saline (PBS, pH 7.4), and ethanol were Sigma-Aldrich, Milan, (Italy)
products. BODIPY FL
disulfide (TCEP) reducing agent were purchased from Thermo Fisher
Scientific, Monza, MB (Italy). cyclo-(Arg-Gly-Asp-D-Phe-Cys)
(c-RGDfC) purchased from Peptides International,
Louisville, Kentucky (USA), sulfur hexafluoride (SF6) from
Rivoira Gas, Rome, RM (Italy), and ICG from Pulsion Medical System,
Munich (Germany) were used without further purification. Water of
Milli-Q purity grade (18.2 MΩ·cm) was produced with an
Elga PureLab Classic, High Wycombe, (UK) deionization apparatus.
6
Differential Scanning Fluorimetry for Protein Stability
7
Assaying Thiol-Dependent Enzyme Activities
8
Recombinant GILT Disulfide Reduction Assay
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Recombinant human GILT expressed in human embryonic kidney (HEK) 293 cells (LS-G50826) was purchased from LifeSpan BioSciences. To confirm reductase bioactivity of the GILT enzyme (see fig. S7, A and B), the disulfide-containing fluorogenic cystine-based reagent BODIPY FL l -cystine (Thermo Fisher Scientific) was diluted to a final concentration of 5 μM in assay buffer (50 mM sodium acetate, 200 μM cysteine, pH 5). Subsequently, 200 ng of GILT, 200 ng of heat inactivated GILT (enzyme heat inactivated at 95°C for 10 min before addition), or no GILT was added to the reaction mixture at a final total volume of 50 μl, and fluorescence liberation was kinetically measured in real time at 37°C for 180 min using a FLUOstar OPTIMA microplate reader (BMG Labtech). Following the subtraction of background fluorescence (samples with 5 μM substrate in assay buffer, no cysteine), rates of disulfide reduction were determined by calculating the slope of the real-time trace between 45 and 90 min (y = mx + c, where y is the relative fluorescence, m is the slope, and x is the time) and made relative to the no GILT control.
9
Fluorescence Assay for Protein Stability
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All chemical compounds were of analytical grade and purchased from Sigma-Aldrich (St. Louis, MO). The fluorescence dyes SYPRO Orange and BODIPY FL-L-cystine were purchased from ThermoFisher Scientific (Waltham, MA). The following microtiter well plates were used: 384-well, black, clear-bottom plates (Nunc International, St. Louis, MO) and hard-shell, thin-wall 384-well PCR plates (Bio-Rad, Hercules, CA). Lyophilized bovine β-lactoglobulin (β-LG) and bovine carbonic anhydrase II were purchased from Sigma-Aldrich (St. Louis, MO).
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