Bacterial membranes harboring penicillin-binding proteins (PBPs) were prepared from LB – carbenicillin (50 μg/ml) cultures grown for 12 h. Uninhibited PBPs were labeled with a fluorescent penicillin (Bocillin FL; Invitrogen, Carlsbad, CA, United States) and quantified by measuring fluorescence intensity as described previously (Zhao et al., 1999 (link)). To observe PBP localization in the bacterial membrane, Bocillin FL-labeled cells were further subjected to a membrane dye, FM-6-64 (Invitrogen; Kocaoglu et al., 2012 (link)) and then observed using a confocal laser scanning microscope (SP8X; Leica) at excitation/emission wavelengths of 488/500–535 and 515/621–678 nm for Bocillin FL and FM-6-64, respectively.
Bocillin fl
Manufactured by Thermo Fisher Scientific
Sourced in United States
Bocillin-FL is a fluorescent penicillin derivative used in the identification and quantification of penicillin-binding proteins (PBPs) in biological samples. It serves as a molecular probe to label and detect PBPs, which play a crucial role in bacterial cell wall synthesis and are important targets for antibiotics.
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Lab products found in correlation
Typhoon laser scanner, by GE Healthcare (1 mentions)
Typhoon 9400 variable mode imager, by GE Healthcare (1 mentions)
Fuji fla 5100 reader, by Fujifilm (1 mentions)
Avibactam, by AstraZeneca (1 mentions)
Chemidoc mp imaging system, by GE Healthcare (1 mentions)
Typhoon fla 9500, by GE Healthcare (1 mentions)
Fb120, by Thermo Fisher Scientific (1 mentions)
Bradford method, by Bio-Rad (1 mentions)
Mini protean tgx, by Bio-Rad (1 mentions)
Amersham imagequant 800 system, by Cytiva (1 mentions)
Avibactam, by MedChemExpress (1 mentions)
Ceftazidime, by Fresenius (1 mentions)
Aztreonam, by Sanofi (1 mentions)
Amhersham imagequant 800, by Cytiva (1 mentions)
Odyssey fc imaging system, by LI COR (1 mentions)
Sds page, by Thermo Fisher Scientific (1 mentions)
Bradford protein assay, by Bio-Rad (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Lsrfortessa flow cytometer, by BD (1 mentions)
Facsdiva software, by BD (1 mentions)
30 protocols using bocillin fl
1
Visualizing Bacterial Penicillin-Binding Proteins
2
Fluorescence Polarisation Binding Assays for PBP3
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Fluorescence polarisation (FP) experiments were carried out as described before [32 (link),35 (link),36 (link),47 (link)]. To determine the binding of Bocillin FL to PBP, 200 nM of PaPBP3 was incubated for 30 min with 100 nM of Bocillin FL (Thermo Fisher Scientific, Waltham, MA, USA). The fluorescence polarisation signal of free Bocillin FL was monitored in a Pherastar microplate reader (BMG Labtech, Ortenberg, Germany) in the polarisation mode with Ex/Em: 485/520 nm excitation and emission filters. Millipolarisation (mP) units were calculated using the MARS data analysis software v30.40.R2 (BMG Labtech, Ortenberg, Germany). All the experiments were done in Sorensen buffer at 37 °C using flat-bottom, black 96-well plates. To determine the IC50 values of the potential Bocillin FL competitors, 5 µL of serial compound dilutions (100 µM to 0.8 µM compound) in 96% DMSO were incubated with PBP3 of P. aeruginosa for 30 min to promote binding of the compounds. Bocillin FL was diluted in Sorensen buffer and added to the reaction and directly measured for 30 min at 37 °C. The FP was continuously measured in the presence and absence of compound and enzyme. The differences between the mP values at 0 min and 17 min were used as the parameter to estimate the IC50 values. The estimated IC50 values were established by sigmoidal nonlinear regression, using Prims v.8 software.
3
Bocillin-FL Assay for PBP6b Activity
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The activity of PBP6b was tested in an assay using the fluorescent penicillin V derivate Bocillin-FL (Life Technologies) (60 (link)). Ten micrograms of PBP6b was incubated with 1 ng/µl of bocillin at 37°C. Reactions were performed in the presence of 10 mM MgCl2 at pH 5.0 (10 mM sodium acetate) and pH 7.5 (10 mM HEPES). Samples were taken after 0, 0.5, 1, 2, 5, 10, 15, 30, and 40 min; the reactions were stopped by addition of 4× SDS-PAGE sample buffer and by boiling for 30 min at 100°C. The proteins were separated by 12% SDS-PAGE before the bocillin-PBP complexes were detected with a GE Healthcare Typhoon laser scanner (excitation laser, 488 nm; emission filter, 520 nm BP 40; photomultiplier tube [PMT] voltage, 600 V), and proteins were subsequently stained with Coomassie blue.
4
PonA1 Labeling in M. smegmatis
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FLAG-tagged isoforms of M. smegmatis PonA1 were produced in TOP10 E. coli. Untransformed TOP10 cells were used as a negative control. Cells were grown overnight in LB and back-diluted 1:100 into 10ml of LB. Once cells reached 0.5 OD600, they were pelleted. Cell pellets were washed once in 1ml of 1x PBS, resuspended in 1ml of 1x PBS, and sonicated twice for 30 seconds each. Bocillin-FL (Life Technologies) was added to the cell solutions at a final concentration of 15 μg/ml and allowed to label protein for 30 minutes at room temperature in the dark. The labeled cell solutions were then pelleted at 100,000 g for 20 minutes to concentrate membrane proteins. The pellets were resuspended in 100 μl of sample buffer containing βME. Proteins were resolved by SDS-PAGE using a 4–15% Tris-Glycine gel. Bocillin-labeled proteins were visualized by imaging with a Typhoon 9400 Variable Mode Imager (GE Healthcare).
5
Fluorescent Labeling of Membrane Proteins
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Portions (100 µg) of membrane proteins were labeled with 100 µM bocillin-FL (Molecular Probes, Thermo Fisher Scientific, Waltham, MA, USA) for 10 min at 30 °C. The reaction was stopped by adding 5-fold-concentrated SDS-PAGE sample buffer (500 mM dithiothreitol, 10% SDS, 250 mM Tris-HCl [pH 6.8], 30% glycerol, 0.02% bromophenol blue). Labeled membrane proteins (20 µg) were separated on a 7.5% SDS-PAGE gel and detected using a 473-nm laser of a Fuji FLA-5100 reader. The quantification of the intensity of the fluorescent bands was performed using ImageJ software. These results are representative of two independent experiments.
6
Fluorescent Penicillin Binding Protein Assay
7
Bocillin FL Labeling of PBPs
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Avibactam was provided by AstraZeneca Pharmaceuticals (Wilmington, NC, USA). Ceftazidime was obtained from Lab Express International (Fairfield, NJ, USA). Oxacillin and amdinocillin were from Sigma-Aldrich Canada (Oakville, ON, Canada). PBPs were labeled using the fluorescent reporter molecule Bocillin FL (Invitrogen-Molecular Probes, Eugene, OR, USA).
8
PBP2a Inhibition Assay in E. coli
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PBP2a was cloned in E.
coli as previously described.26 (link) The assay involves the use of BOCILLIN FL, a commercially
available fluorescent penicillin (Molecular Probes, Inc., CA), as
a labeling reagent for the enzyme active site.30 (link) A typical reaction mixture (20 μL) contains PBP2a
(2.5 μM) and a given concentration of a putative inhibitor (antibiotic)
in 25 mM Hepes, 1 M NaCl (pH 7.0). The mixture is incubated for 5
min at room temperature. At this point, the amount of uncomplexed
(free) PBP is assayed by the addition of BOCILLIN FL to afford a final
concentration of 30 μM, followed by an additional incubation
at 37 °C for 10 min. SDS sample buffer (15 μL) is added
to the reaction mixture, and it is boiled for 3 min. The samples (35 μL
in total) are loaded onto 10% SDS-PAGE, and the gel is developed and
scanned using Storm840 Fluorimager. The fluorescent bands are quantified
using IMAGEQUANT 5.2 software. A control sample is also routinely
prepared in which PBP2a is incubated directly with BOCILLIN FL for
10 min and quenched using the same SDS sample buffer.
coli as previously described.26 (link) The assay involves the use of BOCILLIN FL, a commercially
available fluorescent penicillin (Molecular Probes, Inc., CA), as
a labeling reagent for the enzyme active site.30 (link) A typical reaction mixture (20 μL) contains PBP2a
(2.5 μM) and a given concentration of a putative inhibitor (antibiotic)
in 25 mM Hepes, 1 M NaCl (pH 7.0). The mixture is incubated for 5
min at room temperature. At this point, the amount of uncomplexed
(free) PBP is assayed by the addition of BOCILLIN FL to afford a final
concentration of 30 μM, followed by an additional incubation
at 37 °C for 10 min. SDS sample buffer (15 μL) is added
to the reaction mixture, and it is boiled for 3 min. The samples (35 μL
in total) are loaded onto 10% SDS-PAGE, and the gel is developed and
scanned using Storm840 Fluorimager. The fluorescent bands are quantified
using IMAGEQUANT 5.2 software. A control sample is also routinely
prepared in which PBP2a is incubated directly with BOCILLIN FL for
10 min and quenched using the same SDS sample buffer.
9
PBP Binding Assay Protocol
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PBP binding assays were performed as previously described (30 (link), 31 (link)). Selective labeling of PBP from preincubated samples (10 mg protein) with various concentrations of WYBQ-4 was performed for 15 min, and then 25 μM BOCILLIN FL (Invitrogen) was added, followed by an additional 10 min at 37°C. Proteins were separated by SDS-PAGE, and the PBPs were detected by a gel fluorescence imaging instrument (excitation, 488 nm; emission, 530 nm).
10
Membrane Proteome Labeling Assay
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This method was adopted from a published protocol (62 (link)) with minor modifications. Membrane proteome samples (25 μg in 20 μL PBS) and purified proteins (2.5 μg in 25 μL HEPES pH 7.5 150 mM NaCl) were incubated with 25 μM BocillinFL (Invitrogen) for 20 min at 37°C. Additionally, for the competition assay, purified SaPBP1 was mixed with 2.5 μg (~286 μM final concentration) ampicillin and incubated at 37°C for 10 min prior to the addition of BocillinFL. The reaction was stopped by the addition of 5× SDS-PAGE loading buffer. Membrane proteome was additionally incubated for 10 min at 90°C. The samples were run on a 6 to 20% (wt/vol) SDS-PAGE gradient or 10% (wt/vol) SDS-PAGE gel and visualized using a Bio-Rad ChemiDoc MP imaging system or a GE Typhoon FLA 9500.
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