Hello. Good day! I hope someone can help me or give me an idea on how to prepare 2400 uM cephaloridine using the statement I have read on a published journal.
2.1 Preparation of β-lactamase samples
The methods used were based on those previously described [5]. Cells were grown to mid-log phase, harvested, then re-suspended in 0.067 M NaH2PO4/Na2HPO4 pH 7.3 buffer, supplemented with 0.01 M MgCl2. In this buffer, the permeability of the cells was unaltered over the duration of the experiment (1–2 h). The extent of leakage of β-lactamase into the medium was evaluated by measuring the rate of β-lactam hydrolysis with culture supernatant obtained after centrifugation of the intact cells.
2.2 Determination of I50 values
The concentration of inhibitor necessary to give 50% inhibition (the I50 value) of the hydrolysis of 2400 uM cephaloridine (at 37°C in the above buffer) by whole cell β-lactamase was determined (1200 uM was used for E. cloacae as higher concentrations transiently affected outer membrane permeability). Cephaloridine was selected because all the strains were relatively permeable to it; a marked permeability barrier to the assay substrate can lead to errors when comparing sonicate and whole cell I50 (see Section 2.3).
Dissolve it in the assay buffer to make a stock solution. It is supposedly highly soluble in water. A 10 mM stock solution, for example, would be 4.15 mg/mL.
- Badges
- Science method