In our lab we get lysate samples from all kinds of different labs and one of our go-to clean up steps is to first perform a CME extraction. This involves mixing Chloroform and methanol with the sample in a tube and then letting it separate. The protein forms a wafer at the interface of the two phases. We remove most of the top phase and then clean the pellet with multiple methanol rinses.
We’re trying to figure how we might automate this process in a 2ml square well 96 plate on our Tecan. The idea is that as long as the volumes are the same, the wafer will be at the same level and we can bring a pipette tip down nearly to it. We don’t necessarily need to remove the entire upper phase, just most of it. In the subsequent washes the methanol will dissolve what little chloroform is left and it will be removed.
Has anyone ever tried something like this? Going for the top phase of a two phase mixture with a robot?
we have automated liquid liquid partitioning steps with an aqueous phase and ethyl acetate or cyclohexane using a Hamilton STAR. We use extensive tip mixing with disposable tips to generate a high surface for the partitioning, then we wait ~ 1 min for the phase separation. Centrifugation might help if the phases separation takes too long. The tricky part is, when your upper phase is non-condicting, then you need either pressure liquid level detection (not sure if that is available on a Tecan) or a fixed hight. Aspiration flow rate needs to be slow to not aspirate the lower phase.
Please let me know if you have more specific questions about this procedure!
Hi. If you have a Hamilton system I would record the TADM curves for some samples (just aspirate and dispense to waste). Then look at the curves when the tips enter the protein layer. I would suspet that there will be a shift in the curves. These can be used (in monitoring mode) to trigger a tip exchange and the following aspiration should give you most of the purified proteins.
Hi, these guys did the automated chloroform-methanol extraction on a Biomek FX. You can check it out. I suppose you can contact the authors if you want.