I want to measure the malonyl-CoA in bacteria (not using HPLC). As far as I know, only kit for mouse or human is avaliable. So I just want to know if the "Mouse malonyl-Coa kit" also suits for bacteria as bacteria has cell wall or other factors I don't know.
Thank You!
I have no experience with the specific kit, but: in an ELIZA, you're almost certainly lysing the cells before you put sample onto the kit plate. Looking at the kit description - it relies upon two antibodies specific for malonyl-CoA, which almost certainly bind different regions of the molecule. So the key question isn't about cell walls or the like, but: is your bacterial CoA sufficiently homologous to the mouse CoA that you expect to get a good readout? I assume it's utterly identical, but don't know for certain. And given how small CoA is, even a single atom change might be enough to cause an antibody to not bind.
You could always just develop your own ELIZA assay too - all the kit is is a wellplate coated with a specific antibody, and a second specific antibody with HRP conjugated to it. If you already have specific antibodies you use for Western blotting or the like, you'd just have to do a bit of procedure development to get concentrations of reagents right, and then you could run the assay without having to pay the kit price.
Malonyl-CoA is malonyl-CoA, irrespective of from where it was isolated.
If you were going to measure the enzyme (Acetyl-CoA Carboxylase) that produces it from Acetyl-CoA and Oxaloacetate, that would be an entirely different matter.
The only caveat I see is interference in crude lysates, which you can control by taking some precautionary measures.
You ought to call the tech service people of the reagent supplier.
Best,
It's strange that the one company (MyBiosource) sells separate kits for human, mouse, and rat malonyl-CoA, which is of course the same chemical in all species. What could be the reason?
As for testing a sample of bacteria, you just need to lyse the cells to release the cytoplasm. A physical method such as sonication, bead beater, or French Press is a good way to do it, since they don't introduce any additional chemicals that might affect binding to the first antibody. You may want to do a high-speed spin on the lysate to pellet membranes, cell wall, and large cell fragments, leaving a clear supernatant containing the cytoplasmic constituents.
I do not understand why non-quantitative ELISA is chosen instead of quantitative HPLC method.
ELISA is not the quantitative method at all; i.e., (1) IgG (dimer) is not so specific as compared to IgM (pentamer and/or hexamer) (more than 10-fold increase in specific affinity is expected to be obtainable in IgM as compared to IgG; please see file "The Fascio effect") and (2) photometric assay without prior-purification beforehand gives notorious curved calibration line to give wrong determination values; i.e., c.a. 2.5-fold lower values of fucoidan than HPLC values have been observed, which is due to violence to the "Lambert-Beer law" in the case of the direct photometric assays (please see Fig. 10 in the file; SEC fucoidan determination).
Therefore, I would like to recommend the use of HPLC method.
Dear Sun Tao,
Have u performed the experiment? Can u share the experience?
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