NBD-labeled Spo20-GCC and the mutants (0.3 µM) were incubated at 30°C with liposomes (0.35 mM) of defined radius in HKM buffer supplemented with 1 mM DTT. Emission fluorescence spectra were measured from 520 to 680 nm (bandwidth of 5 nm) upon excitation at 505 nm (bandwidth of 3 nm) in a small quartz cell (total volume 250 µl) in a Shimadzu RF 5301-PC fluorimeter. Control spectra were acquired in the absence of protein to subtract the light scattering signal from the liposomes. For kinetics measurements, NBD fluorescence was continuously measured at 530 nm (bandwidth 5 nm) upon excitation at 505 nm (bandwidth 3 nm). The sample initially contained 350 µM liposomes in HKM buffer (total volume 600 µl) and was placed in a cylindrical quartz cell, which was continuously stirred with a small magnetic bar and thermostated at 30°C. At the indicated times, NBD-labeled Spo20-GCC and PLD were injected from stock solutions through a guide in the cover of the fluorimeter adapted to Hamilton syringes, such as to not interrupt the fluorescence recording.
Rf 5301 pc fluorimeter
Manufactured by Shimadzu
Sourced in United States
The RF 5301-PC fluorimeter is a compact and versatile instrument designed for fluorescence analysis. It features a high-intensity xenon lamp as the excitation light source and a photomultiplier tube as the detector. The instrument provides accurate measurements of fluorescence intensity, wavelength, and lifetime.
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Lab products found in correlation
Methyl β cyclodextrin, by Merck Group (2 mentions)
Tryptophan, by Merck Group (1 mentions)
Xanthurenic acid, by Merck Group (1 mentions)
Rf 10axl fluorescence detector, by Shimadzu (1 mentions)
Uv 1601 pc spectrophotometer, by Shimadzu (1 mentions)
3 hydroxykynurenine, by Merck Group (1 mentions)
Kynurenine, by Merck Group (1 mentions)
9 protocols using rf 5301 pc fluorimeter
1
Membrane Binding Kinetics of Spo20-GCC
2
Liposome Fusion Kinetics Assay
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Experiments were performed in a Shimadzu RF 5301‐PC fluorimeter equipped with a cylindrical quartz cuvette. A suspension (570 µl) of LA liposomes (95 mol% DOPC, 1 mol% NBD‐PE, 1 mol% Rhod‐PE, 3 mol% MPB‐PE, 62.5 µM total lipids final concentration) was incubated with 475 nM pS209 cSTD3 at 37°C under constant stirring in buffer. After 5 min, 30 µl of a suspension of LB liposomes (90 mol% DOPC, 10 mol% DOGS‐NTA‐Ni2+, 62.5 µM total lipids final concentration), pre‐incubated with VAP‐AHis6 (1 µM final concentration), was added. Fusion was measured by recording the NBD‐PE signal at 530 nm (bandwidth 5 nm) upon excitation at 450 nm (bandwidth 5 nm). The percentage of fusion is equal to 100 × ((F − F0)/(Fmax − F0)) where F0 is the signal measured before the addition of LB liposomes decorated with VAP‐AHis6, and Fmax is the signal measured after adding Triton X‐100 (1% v/v final concentration). Liposomes and proteins are injected from stock solutions with Hamilton syringes through a guide in the cover of the fluorimeter.
3
Lipid Transport Assay Using Fluorescent Liposomes
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Experiments were carried out in a Shimadzu RF 5301‐PC fluorimeter equipped with a cylindrical quartz cuvette. A suspension (570 µl) of LA liposomes (62.5 µM total lipids final concentration) made of DOPC and containing 3 mol% MPB‐PE was incubated with 475 nM cSTD3 or pS209 cSTD3 at 37°C under constant stirring in TN3 buffer. After 5 min, 30 µl of a suspension of LB liposomes (77.5 mol% DOPC, 10 mol% DHE, 2.5 mol% DNS‐PE, 10 mol% DOGS‐NTA‐Ni2+, 62.5 µM total lipids final concentration), pre‐incubated or not with VAP‐AHis6 or VAP‐A (KD/MD)His6 (1 µM final concentration) was added. Lipid transport was measured by recording the DNS‐PE signal at 525 nm (bandwidth 10 nm) upon DHE excitation at 310 nm (bandwidth 1.5 nm). The quantity of DHE transported from LB to LA membrane is expressed in terms of mol% DHE in LB liposomes. It is equal to 10 × ((F − F0)/(Fmax − F0)) where Fmax is the signal measured in the absence of pS209 cSTD3 upon the addition of LB liposomes and F0 is the signal measured upon total DHE extraction by 10 mM methyl‐β‐cyclodextrin (Sigma). Liposomes and proteins are injected from stock solutions with Hamilton syringes through a guide in the cover of the fluorimeter.
4
HPLC Analysis of Squid Ommochromes
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Isolated ommochromes from squid were analyzed by high performance liquid chromatography (HPLC) using a Knauer chromatograph (Berlin, Germany) on a Diasphere 120 C18 column (4 × 250 mm; particle size, 5 μm). Solvent A was 10% aqueous acetonitrile containing 0.5% formic acid; solvent B was 100% acetonitrile containing 0.5% formic acid. The pigments were fractionated in a linear gradient (0–40%) of solution B in solution A for 60 min at a flow rate of 0.4 mL/min at 24 °C. Eluted pigments were registered with a Knauer K-2501 UV/Vis detector and an RF-10A-xl fluorescence detector (Shimadzu, Japan).
The ommochromes or standard compounds were dissolved in 100 μL of methanol containing 0.5% HCl. Tryptophan, kynurenine, 3-hydroxykynurenine, and xanthurenic acid (Sigma-Aldrich, Saint Louis, MO, USA) were used as standards. Xanthommatin was synthesized by autooxidation of 3-hydroxykynurenine. Absorption spectra were recorded with a Shimadzu UV–1601PC spectrophotometer. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorimeter. The obtained data were processed with the RFPC version 2.0 software (Shimadzu, Kyoto, Japan).
The ommochromes or standard compounds were dissolved in 100 μL of methanol containing 0.5% HCl. Tryptophan, kynurenine, 3-hydroxykynurenine, and xanthurenic acid (Sigma-Aldrich, Saint Louis, MO, USA) were used as standards. Xanthommatin was synthesized by autooxidation of 3-hydroxykynurenine. Absorption spectra were recorded with a Shimadzu UV–1601PC spectrophotometer. Fluorescence spectra were recorded with a Shimadzu RF-5301PC fluorimeter. The obtained data were processed with the RFPC version 2.0 software (Shimadzu, Kyoto, Japan).
5
Quantifying Promoter Activity in H. salinarum
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To quantify promoter activity, single colonies of H. salinarum strains with different reporter plasmids were inoculated in 5 mL of liquid CM supplemented with 20 μg/mL mevinolin and incubated for 5 days. After pre-growth, cells were diluted to an OD600 ~0.05 in fresh media and grown in standard conditions. At two specific time points (16 and 24 hours), samples (1.8 mL) were taken and the OD600 measured. Sample cells were centrifuged for 5 min at 13,000 rpm and the supernatant was discarded. Cells were then resuspended in 1.8 mL of GFP assay buffer (10mM Tris-HCl, pH 7.5) and mixed vigorously to allow cell lysis. After lysis, cell debris were removed by centrifugation and samples were analyzed in a RF-5301PC fluorimeter (Shimadzu). GFP assays were performed using wavelengths of 488 nm for excitation and 510 nm for emission. Promoter activities were calculated by normalizing the measured fluorescence by the initial cell density (fluorescence/OD600). A control strain of H. salinarum without the reporter plasmid was used to calculate the auto-fluorescence of the cells, and these background values were subtracted from the promoter activities.
6
Fluorescent Dye Encapsulation in Lipid Vesicles
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Solutions (500 μL) containing 2 mM CF in 50 mM Tris-HCl buffer, pH 7.4, or 1 mM PTS in 50 mM Na acetate buffer, pH 5.0, were added to DPPC: LAH films (10 mM total lipid concentration), and the resulting MLVs were extruded.
A Sephadex G25 column (1.5 cm diameter, 45 cm height) was used to separate CF- or PTS-containing vesicles from the non-encapsulated fluorescent probes. The sample (300 μL) was applied to the column, eluted with the desired buffer, and 1 mL fractions were collected. The percentage of CF (or PTS) encapsulation was calculated according toEq. (1) : where Vo and Vi are the excluded and internal column volumes respectively and moles Vo and moles Vi are the number of moles eluted at Vo and Vi, respectively. Fluorescence was measured using a Shimadzu RF-5301PC fluorimeter, with λexc 490 nm, λem 520 nm for CF, and λexc 355 nm, λem 405 nm for PTS [15 (link)]. For the fractions containing vesicles, fluorescence were measured after the addition of 25 μL of an aqueous solution of 10% polidocanol (v/v) to eliminate vesicle-produced light scattering.
A Sephadex G25 column (1.5 cm diameter, 45 cm height) was used to separate CF- or PTS-containing vesicles from the non-encapsulated fluorescent probes. The sample (300 μL) was applied to the column, eluted with the desired buffer, and 1 mL fractions were collected. The percentage of CF (or PTS) encapsulation was calculated according to
7
Kinetics Assays in Fluorimeter
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All kinetics assays were carried out in a Shimadzu RF 5301-PC fluorimeter. The sample (volume 600 μL) was placed in a cylindrical quartz cell, continuously stirred with a small magnetic bar and thermostated. At the indicated time, sample was injected from stock solutions through a guide in the cover of the fluorimeter adapted to Hamilton syringes, such as to not interrupt the fluorescence recording.
8
DOX Uptake Quantification in Cell Cultures
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The cells were
seeded in a 24-well
plate at a density of 4.5 × 105 cells/well and, after
24 h, incubated for 1 h with 1 μM DOX or DOX equivalent amounts
loaded into the βCD-based scaffolds. The fluorescence was measured
after cell lysis with cell culture lysis reagent 1× (Promega,
Madison, USA) at 499 nm (5 nm; excitation maximum wavelength) and
555 nm (10 nm; emission maximum wavelength) with a Shimadzu RF-5301PC
fluorimeter and normalized for protein concentration, measured by
a BCA assay.
seeded in a 24-well
plate at a density of 4.5 × 105 cells/well and, after
24 h, incubated for 1 h with 1 μM DOX or DOX equivalent amounts
loaded into the βCD-based scaffolds. The fluorescence was measured
after cell lysis with cell culture lysis reagent 1× (Promega,
Madison, USA) at 499 nm (5 nm; excitation maximum wavelength) and
555 nm (10 nm; emission maximum wavelength) with a Shimadzu RF-5301PC
fluorimeter and normalized for protein concentration, measured by
a BCA assay.
9
Lipid Transport Assay using Fluorescence
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Experiments were carried out in a Shimadzu RF 5301‐PC fluorimeter equipped with a cylindrical quartz cuvette. A suspension (570 μl) of LA liposomes (62.5 μM total lipids final concentration) made of DOPC and containing 3 mol% MPB‐PE was incubated with 475 nM CSTD3 at 37°C under constant stirring in TN buffer. After 5 min, 30 μl of LB liposomes, containing 10 mol% DHE, 2.5 mol% DNS‐PE, and 2 mol% DOGS‐NTA‐Ni2+ (62.5 μM total lipids final concentration), pre‐incubated or not with VAPHis6 (500 nM final concentration) was added. Lipid transport was measured by recording the DNS‐PE signal at 525 nm (bandwidth 10 nm) upon DHE excitation at 310 nm (bandwidth 1.5 nm). The quantity of DHE transported from LB to LA membrane is expressed in term of mol% DHE in LB liposomes. It is equal to 10 × ((F–F0)/(Fmax‐F0)) where Fmax is the signal measured in the absence of cSTD3 upon the addition of LB liposome and F0 is the signal measured upon total DHE extraction by 10 mM methyl‐β‐cyclodextrin (Sigma). Liposomes and proteins were injected from stock solutions with Hamilton syringes through a guide in the cover of the fluorimeter.
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