β-lactamase activity was measured by hydrolysis of nitrocefin (O’Callaghan et al., 1972 (link); Cavallari et al., 2013 (link); Liu et al., 2016 (link)). Cells were grown to the exponential phase at 30°C with aeration. The cells were then treated with or without Amp or indole for 3 h. Following incubation, 5 ml culture was pelleted, washed with PBS (pH 7.0), and resuspended in the same buffer. Samples were placed on ice and then lysed by sonication, centrifuged, and supernatants were collected. The reaction was initialized by adding cell lysate contained β-lactamase to the reaction mixture containing 25 μg nitrocefin (abcam) and PBS (pH 7.0), and the total volume for each reaction was 1 ml. Enzymatic reaction was performed at 30°C for 10 min, and the change at 486 nm was measured over 1 min on a UV/visible spectrophotometer. The extinction coefficient for degraded nitrocefin was 20.5 mM-1 cm-1. Enzyme activity was measured in nmol min-1 mg-1 nitrocefin hydrolyzed.
Nitrocefin
Manufactured by Abcam
Sourced in United Kingdom, United States
Nitrocefin is a chromogenic cephalosporin compound used as a substrate for the detection of beta-lactamase activity. When hydrolyzed by beta-lactamases, Nitrocefin undergoes a color change from yellow to red, allowing for the rapid identification of beta-lactamase-producing organisms.
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Lab products found in correlation
Spectramax m2, by Molecular Devices (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (1 mentions)
Cefotaxime, by Merck Group (1 mentions)
Kanamycin, by MP Biomedicals (1 mentions)
Ampicillin, by Thermo Fisher Scientific (1 mentions)
Oxacillin, by Merck Group (1 mentions)
Imipenem, by MSD (1 mentions)
Epoch 2, by Agilent Technologies (1 mentions)
Rink amide mbha resin, by Chem-Impex (1 mentions)
9 protocols using nitrocefin
1
Measuring Bacterial β-Lactamase Activity
2
Quantifying Beta-Lactamase Activity
3
Antibiotics Preparation and Characterization
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Kanamycin was purchased from MP Biomedicals; cefotaxime, cephaloridine, and oxacillin from Sigma-Aldrich; cefazolin from Pharmacia & Upjohn SpA; imipenem from MSD; meropenem from Fresenius Kabi NV/SA; ampicillin from Fisher Scientific; amoxicillin from PanPharma; carbenicillin from Pfizer Italy; piperacillin from Lederle/AHP Pharma; temocillin from Eumedica N.V/S.A; and nitrocefin from Abcam.
4
Quantification of Beta-Lactamase Activity
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nitrocefin assay was completed as previously described (7 (link)). Samples in PBS, water, or phenol red-free RPMI-1640 were serially diluted (2-fold) and mixed 1:1 with 0.2 mM nitrocefin (Abcam, Cambridge, UK). Samples were incubated 1–16 h at room temperature and absorbance at 490 nm was read on a SpectraMax M2 plate reader (Molecular Devices, San Jose, California, USA). Data were analyzed with SoftMax Pro software 6.2.2. Recombinant β-Lac standard curves were run simultaneously to quantify β-Lac enzyme concentrations in samples when feasible.
5
Desiccation and Heat Protection Assay
6
Quantification of Bla Activity in Capsules
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The Bla activity was quantified by nitrocefin (Abcam) assay. 5 μL supernatant or 5 μL capsules were diluted into 100 μL using PBS and mixed with substrate nitrocefin (concentration = 50 μM). The resulting absorbance at 490 nm due to the generation of a colored produce was measured as a function of time by a platereader (BioTek Epoch 2). The measurement of enzymatic activity can be explained by the Michaelis–Menten kinetics described in our previous paper27 (link). Experimentally, enzymatic activity is calculated by taking the time derivative of the absorbance at 490 nm in the initial time window, when the absorbance increases linearly with time.
To quantify Bla activity in one capsule, we used the purified T3-Bla to first generate a calibration curve (Supplementary Fig.3a ). We then measured the enzymatic activity for capsules and back-calculated the concentration of Bla in capsules (Ccapsules/μM). Each capsule anchoring Bla was calculated based on .
To quantify Bla activity in one capsule, we used the purified T3-Bla to first generate a calibration curve (Supplementary Fig.
7
Nitrocefin Hydrolysis Assay for β-Lactamase Activity
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The β-lactamase activity was evaluated using a nitrocefin hydrolysis assay [13 (link)]. The cultured cells were centrifuged at 3000×g for 20 min at 4 °C to collect cell-free supernatants. The cell-free supernatants were mixed with 20 μl of 1.5 mM nitrocefin (Biovision, Inc., CA, USA) and incubated at 37 °C. The absorbance was measured every 5 min up to 1 h at 515 nm [14 (link)].
8
Nitrocefin and Clavulanic Acid Characterization
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NMR analysis indicated that the nitrocefin
purchased from BioVision Inc. and Oxoid Limited was significantly
purer than that from Cayman Chemicals. The BioVision nitrocefin was
used in this study. Several values have been reported for the change
in extinction coefficient upon hydrolysis of nitrocefin (e.g., refs (15 (link), 21 (link))). To determine this value independently,
a stock solution containing 5.0 mg of nitrocefin was diluted to a
range of seven concentrations from 10 to 75 μM in 100 mM sodium
phosphate buffer, pH 6.4. The A486 values
before and after complete hydrolysis by 20 min incubation with 5 nM
BlaC were determined. The slope of a linear fit of ΔA486 against nitrocefin concentration yielded
Δε486. The procedure was performed in duplicate,
yielding a Δε486 of 17 ± 1 mM–1 cm–1. Clavulanic acid powder is hygroscopic, so
its concentration was determined by the absorbance at 256 nm in NaOH.
The extinction coefficient was determined by quantitative NMR versus
a standard of trimethylsilylpropanoic acid. For ChemCruz and Matrix
clavulanic acid, which are sold in a cellulose matrix, we found identical
UV–vis spectra, yielding an absorbance at 256 nm of 20.0 ±
0.1 mM–1 cm–1. For TRC clavulanic
acid, which is a pure powder, the UV–vis spectrum is clearly
different, and the NMR spectrum shows impurities. The extinction coefficient
at 256 nm is 18.7 mM–1 cm–1.
purchased from BioVision Inc. and Oxoid Limited was significantly
purer than that from Cayman Chemicals. The BioVision nitrocefin was
used in this study. Several values have been reported for the change
in extinction coefficient upon hydrolysis of nitrocefin (e.g., refs (15 (link), 21 (link))). To determine this value independently,
a stock solution containing 5.0 mg of nitrocefin was diluted to a
range of seven concentrations from 10 to 75 μM in 100 mM sodium
phosphate buffer, pH 6.4. The A486 values
before and after complete hydrolysis by 20 min incubation with 5 nM
BlaC were determined. The slope of a linear fit of ΔA486 against nitrocefin concentration yielded
Δε486. The procedure was performed in duplicate,
yielding a Δε486 of 17 ± 1 mM–1 cm–1. Clavulanic acid powder is hygroscopic, so
its concentration was determined by the absorbance at 256 nm in NaOH.
The extinction coefficient was determined by quantitative NMR versus
a standard of trimethylsilylpropanoic acid. For ChemCruz and Matrix
clavulanic acid, which are sold in a cellulose matrix, we found identical
UV–vis spectra, yielding an absorbance at 256 nm of 20.0 ±
0.1 mM–1 cm–1. For TRC clavulanic
acid, which is a pure powder, the UV–vis spectrum is clearly
different, and the NMR spectrum shows impurities. The extinction coefficient
at 256 nm is 18.7 mM–1 cm–1.
9
Peptide Synthesis and Membrane Protein Assays
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Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Chem-Impex Int’l., Wood Dale, IL, USA), ortho-Nitrophenyl-β-galactoside (ONPG) (Research Products International Co., Mt Prospect, IL, USA), and nitrocefin (Biovision, Milpitas, CA, USA) were stored as powders at −20 °C and prepared fresh for each use. Fmoc-protected amino acids were purchased from GL Biochem Shanghai Ltd. China and Rink Amide MBHA resin from Chem-Impex. Trypsin powder was obtained from Amresco (Solon, OH, USA). Solid POPC lipid (>99% purity) was purchased from Avanti Polar Lipids (Alabaster, AL, USA). Non-natural amino acid Fmoc-β-(1-Azulenyl)-L-Alanine was synthesized according to previously published protocols [23 (link),25 (link)].
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