Fluorescent conjugates were constructed using Alexa Fluor 488 5-TFP (Life Technologies, Eugene, OR, USA) following the manufacturers protocol and methodology previously described in [15 (link)]. Saporin SO6 or OKT10-SAPORIN were added to 100 µL carbonate buffer (1 M NaHCO3, pH 9.0) and 100 µL of Alexa Fluor 488 5-TFP (10 mg/mL in DMSO) and stirred for one hour at room temperature. Unconjugated fluorophore was removed by dialysis against PBS at 4 °C. The Beer–Lambert law was used to determine the concentrations of the fluorescent conjugates by measuring the absorbance of the samples at 280 and 495 nm using a Hitachi U1100 Spectrophotometer.
Alexa fluor 488 5 tfp
Manufactured by Thermo Fisher Scientific
Sourced in United States
Alexa Fluor 488 5-TFP is a fluorescent dye used for labeling proteins and other biomolecules. It has an excitation maximum at 488 nm and an emission maximum at 519 nm, making it suitable for detection in flow cytometry and fluorescence microscopy applications.
Automatically generated - may contain errors
Lab products found in correlation
U 1100 spectrophotometer, by Hitachi (2 mentions)
Cytation 5 plate reader, by Agilent Technologies (1 mentions)
Prism 8, by GraphPad (1 mentions)
Fluoromax 4 spectrofluorometer, by Horiba (1 mentions)
Zeba desalting column, by Thermo Fisher Scientific (1 mentions)
Pacific blue succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Pacific orange succinimidyl ester, by Thermo Fisher Scientific (1 mentions)
Phosflow perm buffer 3, by BD (1 mentions)
Phosflow fix buffer 1, by BD (1 mentions)
7 protocols using alexa fluor 488 5 tfp
1
Fluorescent Conjugate Formation Protocol
2
Fluorescent Labeling of Saporin and OKT10-SAP
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To detect the trafficking of internalised saporin and OKT10-SAP together with their proposed endolysosomal escape in the presence of SA, fluorescent conjugates were constructed with an Alexa Fluor 488 5-TFP (Life Technologies, OR, USA). This was achieved by adding 800 μl of 9.3 mg/ml saporin SO6 or 3.5 mg/ml OKT10-SAPORIN to 100 μl carbonate buffer (1 M NaHCO3, pH 9.0) and 100 μl of Alexa Fluor 488 5-TFP (10 mg/ml in DMSO). Following stirring for 1 h at room temperature to effect conjugation, unconjugated fluorophore was removed by exhaustive dialysis for two hours at 4°C against 2 L PBS followed by a further 2 L of PBS overnight at 4°C. The concentrations of the resultant fluorescent conjugates were calculated using the Beer-Lambert law from their absorbance at 280 nm and 495 nm as measured on a Hitachi U1100 Spectrophotometer.
3
Quantifying protein-protein interactions
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Fluorescence spectroscopy was used to quantify affinities of protein-protein and protein-peptide interactions by fluorescence (de) quenching. GST-EF3L (Gcn1 1331-1670 ) labeling by Alexa Fluor 488 5-TFP (Life Technologies) was performed as described previously (Janovi c et al., 2019) . Measurements of EF3L interactions with Gcn20 variants were performed at room temperature in phosphate-buffered saline (pH 7.4) on a FluoroMax-4 spectrofluorometer (Horiba), monitoring fluorescence intensity at an excitation wavelength of 489 nm and an emission wavelength of 515 nm. The slit width was 10 nm and the integration time was 1 s. Peptides PTDDIAEALGELSLKKKKKKTK and PTDDIAEALGEL[pSer]LKKKKKKTK corresponding to Sui3 68-89 were N-terminally conjugated with fluorescein isothiocyanate over 6-aminohexanoic acid (GenScript), and tested for GST-Tif5 and GST-Gcd6 binding at room temperature in assay buffer (30 mM HEPES [pH = 7.5], 100 mM KCl, 0.1 mM MgCl 2 , 10% glycerol, 1 mM DTT, 0.1 mM PMSF, 0.5 3 PhosSTOP) on a Cytation 5 plate reader (BioTek), monitoring fluorescence intensity at an excitation wavelength of 485 nm and an emission wavelength of 528 nm. Kds were calculated by Prism 8 software (Graphpad) using nonlinear asymmetric sigmoidal regression (5PL). The experiments were performed in triplicates.
4
Fluorescent Adenovirus Labeling Protocol
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After purification, adenovirus particles were incubated with a 20-fold molar excess of chemically reactive Alexa Fluor 488 5-TFP (A30005 , Thermo Fisher Scientific, USA) for 2 h in the dark at room temperature with gently stirring in 10% glycerol in PBS with 100 nM sodium bicarbonate, pH 7.2. Unbound dye was removed from the labeled virus using Zeba desalting columns (cat. 89889, Thermo Fisher Scientific, USA) with exchange buffer 10% glycerol in PBS according to the manufacturer's protocol. Flow through aliquots containing labeled virus (HAdV-AF488) were stored at −80°C.
5
Fluorescent Labeling and Imaging of Vibriophages in Bacterial Biofilms
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Bacteriophages were labelled as described previously37 (link). Briefly, 100 μL of purified vibriophages with a concentration of 1012 plaque forming units (PFU) per mL were mixed with sodium carbonate (0.1 M final) and then incubated with 0.1 mg Alexa Fluor 488 5-TFP (A30005, Thermo Fisher Scientific) for 1 h at room temperature under continuous shaking. The reaction mixture was first dialyzed at 4 °C against 500 mL of phosphate-buffered saline (PBS) for 3 hours and then dialyzed overnight at 4 °C against 2 L of PBS. This was done to separate phages from unbound dye. This stock of fluorescently labelled phages was stored at 4 °C.
Biofilms of the mRuby3-expressing V. cholerae WT strain (KDV657) were grown for 24 h, before the inflowing M9 medium was exchanged to contain tetracycline for 6 h and fluorescently labelled phages (a 1:100 dilution of the fluorescently labelled phage stock). As a control treatment, M9 medium without tetracycline, but containing the same concentration of vibriophages was added for 6 h. Biofilms were imaged for the 6 h of phage exposure with a time resolution of 2 h (Extended Data Fig. 9c ).
Biofilms of the mRuby3-expressing V. cholerae WT strain (KDV657) were grown for 24 h, before the inflowing M9 medium was exchanged to contain tetracycline for 6 h and fluorescently labelled phages (a 1:100 dilution of the fluorescently labelled phage stock). As a control treatment, M9 medium without tetracycline, but containing the same concentration of vibriophages was added for 6 h. Biofilms were imaged for the 6 h of phage exposure with a time resolution of 2 h (
6
Single Cell Phospho-Flow Cytometry
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Single cell signaling analysis was carried out using phospho flow with fluorescent cell barcoding as described19 . Briefly, B cells were treated as described in the figure legends before fixation (BD Phosflow Fix Buffer I, BD Biosciences, Franklin Lakes, NJ, USA). The cells were then washed three times with PBS and stained with different concentrations of the barcoding reagents Alexa Fluor 488 5-TFP, Pacific Blue Succinimidyl Ester and Pacific Orange Succinimidyl Ester (all from ThermoFisher Scientific, Waltham, MA, USA) for 20 min at room temperature. The cells were then washed twice with flow wash (PBS, 10% FCS and 0.09% sodium azide), combined in one tube and permeabilized with BD Phosflow Perm Buffer III (BD Biosciences) which was pre-cooled at −20 °C, and kept at −80 °C until further processing. Before antibody staining, the cells were washed three times with flow wash. The antibodies used in this study are listed in Supplementary Table 1 , including the surface marker anti-CD19. After 30 min incubation with the antibodies, the cells were washed once, resuspended in flow wash and analyzed with a BD FACSCanto II (4-2-2) cytometer equipped with 405 nm, 488 nm and 633 nm lasers. The data were analyzed in Cytobank (https://cellmass.cytobank.org/cytobank/ ) as described4 (link).
7
Fluorescent Labeling and Imaging of Vibriophages in Bacterial Biofilms
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