TB_KKG6K and PCγC-terminal were synthesized on solid phase, using standard protocols for the 9-fluoroenylmethoxy chemistry and were then purified by reversed-phase high-performance liquid chromatography (RP-HPLC) and analyzed by electrospray ionization-mass spectrometry (ESI-MS) as described previously (7 (link), 14 (link)). For fluorescence-based analyses, the TB analog was synthesized and conjugated to the 6-aminohexanoic acid linker first and then to the green fluorophore FITC as described elsewhere (77 (link)). The FITC-conjugated peptide (FITC-TB_KKG6K) was purified by RP-HPLC on a Jupiter 10-μm Proteo 90A° (100 × 21, 20 mm) column, with a flow rate 20.0 mL/min, and analyzed by ESI-MS as described by Kakar et al. (7 (link)): calculated mass (Da), 2221.28; found, 1111.88 [M + 2H]2+; 741.82 [M + 3H]3+. The P. chrysogenum antifungal protein PAFB was expressed using a P. chrysogenum -based expression system (78 (link)) and purified by cation-exchange chromatography as previously described (32 (link)). For fluorescence-based analyses, PAFB was labeled with the green fluorophore BODIPY FL EDA (Invitrogen, Waltham, MA, USA) as described previously (32 (link)).
Bodipy fl eda
Manufactured by Thermo Fisher Scientific
Sourced in United States
BODIPY-FL-EDA is a fluorescent dye product offered by Thermo Fisher Scientific. It is a member of the BODIPY dye family, known for their bright fluorescence and photostability. The core function of this product is to serve as a fluorescent label or probe for various biological and chemical applications.
Automatically generated - may contain errors
Lab products found in correlation
Sodium chloride (nacl), by Merck Group (2 mentions)
Potassium chloride (kcl), by Merck Group (2 mentions)
Potassium phosphate monobasic, by Merck Group (2 mentions)
Sulfo nhs, by Thermo Fisher Scientific (1 mentions)
Pd 10 desalting column, by Cytiva (1 mentions)
Bca protein assay, by Thermo Fisher Scientific (1 mentions)
Foetal bovine serum fbs, by Thermo Fisher Scientific (1 mentions)
Sucrose, by Merck Group (1 mentions)
Sodium phosphate dibasic, by Merck Group (1 mentions)
Jeffamine m 1000, by Huntsman (1 mentions)
9 protocols using bodipy fl eda
1
Synthesis and Purification of Fluorescent Peptides and Proteins
2
Fluorescent Labeling of Antifungal Protein
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PeAfpA was labelled with the green fluorophore BODIPY™-FL EDA (4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine, Invitrogen, Thermo Fisher Scientific) as described with minor changes (Sonderegger et al. 2017 (link)). In brief, 3-mg lyophilized PeAfpA were dissolved in 1 mL of 0.1 M MES (2-(N-morpholino)ethanesulfonic acid) buffer pH 4.5. Subsequently, a reaction mixture containing 200 μL of PeAfpA, 30 µL of 0.1 M EDAC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride, Invitrogen), 15 µL of 100 mM Sulfo-NHS (N-hydroxysulfosuccinimide, Invitrogen) and 30 µL of 50 mM BODIPY in MES buffer was incubated in darkness at 25 °C, with gently mixing for 3 h. The reaction mixture was dialyzed (3.5 kDa molecular weight cut-off; Thermo Scientific, Thermo Fisher Scientific) several times against deionized water at 4 °C to remove free BODIPY and salts. Labelling efficiency and protein concentration were determined spectrophotometrically. Labelled protein retained full antifungal activity (Supplemental Fig. S1 ).
3
Conjugation of CpoBD13 with BODIPY FL EDA
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Lyophilised CpoBD13 (2 mg) was resuspended in 900 µL of conjugation buffer (100 mM MES, 500 mM NaCl, pH 6.0) followed by the addition of 0.71 mg 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 2 mg N-hydroxysuccinimide (50 µL of each, dissolved in conjugation buffer). After incubating at RT for 15 min, the pH of the reaction was adjusted to between 7–8 by the addition of 20× PBS. BODIPY FL EDA (Thermo Fisher Scientific) was resuspended in dimethylsulfoxide to a concentration of 20 mg/mL before it was subsequently added to the reaction mixture (41 µL, molar excess of 5:1). After incubating the reaction for 150 min, salts and unbound BODIPY were removed by using a PD-10 desalting column (Cytiva, Marlborough, MA) as per the manufacturer’s instructions.
4
BODIPY-Labeled Polymer Synthesis and Characterization
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Man-PRX (100 mg, 2.01 μmol PRX, 53.6 μmol threaded α-CDs in PRX) and CDI (38.2 mg, 236 μmol) were dissolved in dehydrated DMSO (5 mL) under a nitrogen atmosphere and the solution was stirred for 6 h at room temperature. 4,4-Difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionyl ethylenediamine hydrochloride (BODIPY FL EDA; Thermo Fisher Scientific, Waltham, MA, USA) (1.02 mg, 2.75 μmol) was added to the reaction mixture, and the solution was stirred for 24 h at room temperature, with protection from light. After the reaction, the resulting polymer was purified by dialysis against water for 3 days using Spectra/Por 4 (MWCO: 12,000–14,000), and the recovered solution was freeze-dried to yield BODIPY-labeled Man-PRX (BODIPY-Man-PRX) (99.4 mg). The number of BODIPY molecules modified onto Man-PRX was determined by measuring the absorbance at 503 nm (one BODIPY molecule was modified onto 6.3 Man-PRX molecules). BODIPY-labeled HEE-PRX (BODIPY-HEE-PRX) was synthesized and characterized in the same manner. In subsequent experiments, unlabeled and BODIPY-labeled PRXs were mixed to adjust the fluorescence intensities of BODIPY-Man-PRX and BODIPY-HEE-PRX.
5
Fluorescent Protein Labeling Protocol
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The proteins were labelled with the green fluorophore BODIPY FL EDA (Life Technologies) as described with minor changes [26 (link)]. In brief, protein samples (0.4 mM) were dissolved in 0.1 M MES buffer pH = 4.5. BODIPY was added to a final concentration of 10 mM and subsequently EDAC (Life Technologies) and Sulfo-NHS (Life Technologies) were added to a final concentration of 10 mM and 5 mM, respectively. The reaction mixture was stirred in darkness for 3 h at 25°C, followed by dialysis against ddH2O. Protein concentration and degree of labelling were determined spectrophotometrically.
6
Fluorescent Protein Labeling Protocol
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Proteins were labeled via carboxyl groups with the fluorophore BODIPY-FL-EDA (Life Technologies), as previously described (51 (link), 60 (link)). Unbound BODIPY and salts were removed through dialysis using benzoylated cellulose tubes (2,000 molecular weight cutoff; Sigma) against deionized water. Protein concentration was determined by UV spectrometry.
7
Purification and Labeling of NaD1 Antimicrobial Peptide
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NaD1 was purified from the flowers of Nicotiana alata as described in van der Weerden et al. (2008) [12 (link)]. In brief, flowers were crushed in a mortar and pestle with liquid nitrogen and then subjected to an acid and heat treatment. Protein was then purified using cation-exchange chromatography and reverse-phase high-performance liquid chromatography (RP-HPLC). The protein concentration was determined using the bicinchoninic acid (BCA) protein assay (ThermoFisher, Scoresby, Australia). NaD1 was fluorescently labelled with 4,4-Difluoro-5,7-Dimethyl-4-Bora-3a,4a-Diaza-s-Indacene-3-Propionyl Ethylenediamine, Hydrochloride (BODIPY-FL-EDA, Life Technologies, Carlsbad, CA, USA) as described in [13 (link)]. The LL-37 peptide (amino acid sequence: LLGDFFRKSKEKIGKEFKRIVQRIKDFLRNLVPRTES) was synthesised by GenScript (Piscataway, NJ, USA).
8
Synthesis and Characterization of Functionalized Nanoparticles
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Yellow-green carboxylate-modified polystyrene NPs of 100 nm and 20 nm nominal diameter were purchased from Invitrogen (PS-COOH100 and PS-COOH20). PS-COOH100 NPs were pegylated through EDAC chemistry using Jeffamine M1000 (Hunts-man) and purified by centrifugal washings (3 × 30 minutes at 17 000 rcf, 20 °C) (PS-PEG). Bare 50 nm silica NPs were purchased from Kisker (SiO 2 ). Carboxylated Fe 3 O 4 NPs were synthesized following the protocol of Sun et al. 38 and coated by poly(maleic)-alt-1-octadecene (Sigma) according to Lin et al. 39 obtaining NPs of about 50 nm in hydrodynamic diameter. For cell uptake studies, Fe 3 O 4 NPs were fluorescently labelled with BODIPY FLEDA (Lifetechnologies) which was attached to the NP surface by EDAC chemistry. Foetal bovine serum (FBS) was purchased from Fisher. Sucrose, sodium phosphate dibasic, potassium phosphate monobasic, sodium chloride and potassium chloride are from Sigma.
9
Fluorescent Carboxylated Fe3O4 NP Synthesis
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Carboxylated Fe 3 O 4 NPs were synthesized following the protocol of Sun et al. (2003) and coated by poly (maleic)-alt-1octadecene (PMAO, Sigma) according to Lin et al. (2008) for water trans-fer. Sucrose density gradient ultracentrifugation (6.6-66 w/ w%, 187k rcf, 4 h, 20 • C) was performed to purify them from excess polymer. Purified NPs were fluorescently labelled by BODIPY ® FLEDA (LifeTechnologies) through N-(3-dimethylaminopropyl)-Nethylcarbodiimide hydrochloride chemistry (Sigma). This dye was chosen because, according to the supplier, its quantum yield is min-imally affected by the pH and polarity of the solvent (Karolin et al., 1994) . Unbound free dye was removed by Amicon centrifugal filters (50 kDa MWCO, 360 rcf). Sucrose, sodium phosphate dibasic, potassium phosphate monobasic, sodium chloride and potassium chloride are from SIGMA.
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