Currently, I'm trying to follow the iodometric method from the following paper: Article Iodometric Assay Method for Beta-Lactamase with Various Beta...
However, it's not very clear and I need some help with a better protocol for the same. I would hence request some guidance with this.
If you have a UV spectrophotometer you can just measure directly. Nitrocefin is nice because the absorption peak is in the visible light range but Penicillin G (and I think also ampicillin) for example has a peak around 320. You can monitor the disappearance of that peak as your assay.
Viet Thanh Nguyen
the original question was looking for an assay other than nitrocefin, and the kit you mention is in fact a nitrocefin based assay.
Thank you Michael Sir and Viet Sir. However, I've linked my question with a research paper. What I'm really not clear about is the enzymatic hydrolysis section in it. It would be great if some light could be shed on it.
Harsha, it is not very clear what your question is. What difficulty are you having?
Dear Harsha
The paper you have mentioned is too old and your question is not clear. There should be either two objectives with beta-lactamase experiment. Firstly the kinetic assay with different beta-lactam antibiotics including the chromogenic cephalosporin nitrocefin or only the beta-lactamase activity assay. Nitrocefin is very good for only the activity assay. However, to determine the kinetic properties like-Km or Kcat value, nitrocefin is not a good choice as currently, I am working with it and still in dark.
Many thanks
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