My lab has employed a FRET-based nucleic acid cleavage assay with a fluorescein labelled RNA bound to a DNA with black hole quencher. Historically, we would always get picture-perfect hyperbolic reaction progress curves. However, for the past few months, we have been having problems with purification of one of the ribonuclease enzymes we study. Specifically, there is a 5-10 minute "lag phase" that occurs prior to the reaction start, as well as one or more "glitches" in the reaction where it slows down and then speeds back up. We purify other RNase Hs using the same reagents and protocol, and these enzymes do not have the same issues. The issues are more prominent as substrate concentration increases. Additionally, measurement of the Km of current batches of this enzyme is 3-4 fold higher than it was historically. Additionally, this enzyme is now coming through SEC mostly as a high MW aggregate, whereas it used to be all monomeric. Nothing to our perception has changed in our procedure, so it is probably something quite subtle. Any help is appreciated!
It seems likely that the problem is with the expression/purification, rather than the assay. Check all the reagents, solutions, and columns used in the procedures.
FYI, just wanted to update that we have solved this issue mostly. The aberrant behavior seemed to be related to nucleic acid contamination carryover from the protein purification. After implementing a benzonase treatment (promiscuous nuclease) during induced cell lysis and a high salt wash during purification, the problem mostly went away, as evidence of NA contam by UV-Vis and EtBr agarose.
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