I am working on quorum quenching enzyme assay. My AHL stock is prepared in DMSO. In CV026 bioassay, my enzyme shows quorum quenching activity. For kinetic study, we are performing OPA based assay for detection of HSL. The read out of the OPA assay is very less. As the substrate concentration increases,the DMSO % goes upto 6-7 % in the final reaction mixture of the enzyme and the AHL (in DMSO). Is there any other way to prepare a stock solution with less DMSO without solubility issue ? Any advice on improving the OPA assay is requested.
I just found your posting by chance today. Although your question is quite long ago, I would like to answer it as no one did this before. You might simply prepare stocks with much higher AHL concentrations. What is your currently used concentration? In my paper I wrote: "Stock solutions of C6-HSL and C8-HSL (400 mM) were prepared in anhydrous dimethyl sulfoxide (DMSO) containing 0.2% glacial acetic acid and stored in aliquots at -20°C." Even when I used 2 mM AHL for biocatalysis, the DMSO concentration was negligible. For long-chain AHLs you might have to use lower stock concentrations. The activity assay I published might be an alternative to the OPA method if it is not working properly. Good luck for your research!
Article Fast, Continuous, and High-Throughput (Bio)Chemical Activity...
Thank you so much Daniel for answering my query. I also prepared the stock in DMSO, around 20 mM stock concentration. 400 mM stock is better as finally we can get very less volume of DMSO upon dilutions for different experiments. Is addition of 0.2% glacial acetic acid done for stabilization of the signal molecules in acidic condition ?
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