Trouble with a citrate synthase assay

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Filigree
Filigree's picture
Trouble with a citrate synthase assay

Hi, everybody.

I'm an undergraduate working in a cardiophysiology lab, and my assigned project is to measure citrate synthase activity in rat soleus muscles. Because of our budget, we aren't using a kit for the assay, instead, we're conducting the assay by a protocol that was sent to us by a colleague at Mizzou similar to the one included with the kit.

The problems with our results thus far are severalfold - one, that our numbers are in the neighborhood of 50% lower than the expected values included with this protocol, and two, that between groups, our numbers are the reverse of what they should be (exercise groups, for example, are lower than control groups, which doesn't make sense).

For the first problem, we've been checking our reagents and suspect that one of them (DTNB, the substrate that CoA-SH acts on to produce the yellow compound we detect spectrophotometrically) was stored improperly and as such may havve skewed our results downward. The other possibility is that our reagents are too cold - citrate synthase operates at body temperature, but we've been directed to keep everything on ice, so I'm not sure what' the right way to do it.

As for the second problem, I have no idea what's causing the reversed results. I would suspect error on my part, but in that case, I'd expect the results to be more scattered and not showing any particular pattern. Instead, the results are consistently flipped - ALL the ones that are supposed to be high are low and vice versa.

I'm attaching the protocol that we used - it's modified a bit from the one we recieved, with the difference being that we centrifuged our samples after homogenizing and sonicating them. We know that the centrifuging didn't affect our activity levels, because numbers are similar before and after we started centrifuging - the only difference being that our percent error is much lower now.

Apologies for the length and complexity. It seems like there's not much help to be found on this sort of thing just floating around on the internet, and this isn't an assay our lab is familiar with, so I thought I'd give you all a shot.