The study is currently working on the binding properties of antibacterial peptide to plasmid DNA of Pseudomonas aeruginosa after the discovery that this particular ABP (known to have membrane permealising property, shown initial increase in fluorescence signal) displayed an eventual declined trend of fluorescence signal using the SYTOX Green uptake assay (the proposed reasons are competitive binding between intercalating SYTOX Green stain and the ABP to bacterial DNA; and DNA degradation due to the permealised membrane). Many studies have suggested the isolation using alkaline lysis method (especially commercial kits), SDS lysis method etc., however, I was restrainted from using them due to certain reasons. Is it possible to conduct gel retardation assay without manually isolating plasmid DNA from the bacteria? Or there could be another assay that will lead to it, practically? Thank you very much for pondering or answering my question.
Unless you think the ABP specifically binds to plasmids only from P. aeruginosa (because of high GC content?), any plasmid, and potentially any double-stranded DNA, could be used to test for competition with SYTOX Green. You could buy plasmid, use sheared calf thymus or salmon sperm DNA, or use double-stranded synthetic oligos. It might be interesting to use oligos, since then you could explore the effect of nucleotide composition on the binding of the ABP.
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