Talk:MiPNet17.04 CitrateSynthase

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Difficulties with CS in microplate reader

My question concerns the Citrate Synthase activity assay described in MiPNet17.04. We had tried to reproduce an assay according to instructions for a commercially available kit from Sigma. Due to proprietary components ("Assay Buffer") we were forced to construct a hybrid method, mainly trying to replace the Sigma kit's "Assay buffer". This has not worked out for us - we were getting magnitudes of order lower enzyme activcities just using the Sigma Citrate Synthase enzyme as positive control for assay conditions. Sigma tech support was unable to help us, mainly due to their proprietary constraints.

My search for alternative assays brought me to the MiPNet publication 17.04, which we tried to adapt to our conditions: I should mention that we are forced to deviate from the MiPNet protocol in several aspects, usage of a microplate reader instead of a traditional spectrophotometer, including downsizing of volumes with maintenance of the final concentrations of components; OAA dissolved in the Tris buffer instead of TEA buffer (we later tried TEA buffer as well with no better results), a 30 mM stock of Acetyl CoA in water instead of 12.2 mM (final concentrations in assay are same), DTNB dissolved in absolute ethanol. So, quite a few changes. Having done that, we determined and calculated activity of the commercial citrate synthase enzyme (Sigma, as recommended) with much, much lower than expected activities based on Sigma's lot information.

Stephanie Wohlgemuth - 2012-06-26

General comments

I am not surprised that the multiwell assay approach yields much lower and quite irreproducible results. This expectation is derived from an extensive study in my lab comparing quartz glass cuvettes with dischargeable plastic cuvettes. These were 1 ml cuvettes, and in microplates the surface to volume ratio is tremendously higher. Consistently, CS and LDH activities were significantly lower in plastic compared to glass (CS and LDH are VERY VERY different assays!). We hoped to obtain a correction factor to be applied to a series of experiments run with plastic cuvettes, but the reproducibility was entirely insufficient: we obtained 40% to 85% in plastic compared to glass. This was not due to different brands of plastic cuvettes – we tested a variety of suppliers, obtaining a similar range of deviations, with highly reproducible activities in the glass cuvettes. But factorial analysis failed when addressing the question as to the cause of the inactivation in plastic cuvettes. Starting the assay in the plastic cuvette and transfer of the sample into glass for measurement should have deactivated the enzymes. But this was not observed. What remained from this lengthy test procedure was (1) the note in the MiPNet 17.04 protocol to use glass, and (2) discussions with several highly experienced ‘classical’ biochemistry groups, all of whom confirmed: ‘never use plastic if you want to do science’.

Another very general note: Small variations of a protocol may result in big effects, the cause of which is frequently not understood. E.g. in the CI assay, replacing cyanide+acide with just one of the inhibitors does not work. This must have been checked many times before, but I wanted to simplify the assay – without any luck. Thus any of the variations you describe may need testing, without which we may not be able to provide even an educated guess.

Erich Gnaiger - 2012-06-26