Beta-Lactamase Activity Assay Kit

Protocol tips

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		Measure the absorbance (A490) in a microplate reader in kinetic mode for 30–60 minutes at room temperature.

Downstream tips
Measure the absorbance (A490) in a microplate reader in kinetic mode for 30–60 minutes at room temperature.

Publication protocol

We explored differences in β-lactamase activity by using a β-lactamase activity assay kit (Sigma-Aldrich, St. Louis, MO, USA). This assay is based on the hydrolysis of the chromogenic molecule nitrocefin, a non-antimicrobial cephalosporin, by β-lactamase which leads to the production of a colorimetric product. Formation of this product is monitored by measuring absorbance at 490 nm in an Envision multilable plate reader; every minute, for 60 to 90 min at 25 °C. The amount of enzyme required to hydrolyze 1.0 μmol of nitrocefin per minute at pH 7.0 at 25 °C is equal to one unit of β-lactamase [10, 11, 24]. Bacterial cultures were grown in 96-well microtitre plates in the presence or absence of antibiotics. Antibiotics were added to the microtitre plates at concentrations of ¼ MIC and the β-lactamase inhibitors SUL, TAZ and AVI were added at 4 mg/L. Planktonic cultures were grown overnight in LB broth at 37 °C, then adjusted with double-concentrated MHB to obtain a final inoculum of 5 x 105 cfu/ml in the 96-well plates. For every condition 10 wells in the same plate were filled and the plates were incubated at 37 °C for 24 h. After incubation, the content of the 10 wells was collected in a pre-weighed plastic tube and centrifuged at 10 000 RCF for 10 min. Then, supernatant was discarded and the pellet was weighed and resuspended with 5 μL of β-lactamase assay buffer per mg sample. Subsequently, samples were sonicated for 5 min, placed on ice for 5 min and centrifuged at 16 000 RCF at 4 °C for 20 min. 1 – 50 μL of the unknown samples was added to a clear flat 96-well plate and supplemented with nitrocefin and buffer to a final volume of 100 μL. Immediately after addition of nitrocefin, absorbance at 490 nm was measured in an Envision plate reader.

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Manufacturer protocol

Download the product protocol from Sigma-Aldrich for Beta-Lactamase Activity Assay Kit below.

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