I'm studying the interaction between DNA (pBR322 plasmid) and a synthetic antibacterial peptide using gel retardation assay/electrophoretic mobility shift assay (EMSA). The plasmid is set fixed at 10 microg/mL, 2.5 microL into each well. However, I'm not sure about the concentration of plasmid (increasing across each well) I should use to treat the plasmid prior to loading them into each well. I've been referring to many papers using the same assay, each suggested different titres for all different peptides, for instance, 2.5, 5, 10, 20, 40, 80, 160 microg/mL OR
4, 8, 12,16, 20, 30, 40, 60 microg/mL OR
0.5, 2.5, 5, 25 microM OR
0.9, 1.8, 9, 18, 90, 180 microg/mL.
Is there a way to determine the best suitable titre for my designed peptide?
The best range of concentrations depends on the affinity. The lower the affinity, the higher the concentrations needed. You could do a preliminary experiment using 10-fold serial dilutions (such as 200, 20, 2, 0.2 and 0 µg/ml) in order to determine the right range, then follow up with a set of 2-fold serial dilutions to cover that range thoroughly.
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