Using Malate as a substrate I am unable to get a linear graph (Increase in optical density at 340 nm) when I am performing Malic enzyme assay in a time course mode. What should be the optimum concentration of the Malate during the reaction (Presently it's 10 uM) and what should be the optimum concentration of MgCl2? If any one has the protocol for it, kindly share it with me.
Hi Deepti,
Did I get it correct, that you have in your assay only 10 µM of malate?
If yes, then, increase [malate] up to 2,5 mM (in some assays also 5 mM is used).
I am really thankful for your kind suggestion Alex.... Please suggest the concentration of MgCl2 as well if you use the same in your assay
Hi Deepti,
I would suggest to try MnCl2 2 mM or also MgCl2 2 mM (to see what is the best), Malic acid (malate) 2,5 mM, NAD+ and NADP+ 0,4 mM (I do not know which one is preferred by your enzyme (again, you may try what is more speedy). If you measure Abs at 340 nm - it is recommended to quartz cuvettes. If you have glass cuvettes - it is also possible to measure at 360 nm.
I hope it helps and
Good luck!
Thanks a lot Alexnder I would try to use the concentrations you have mentioned in.... We have quartz cuvettes and UVettes both so we can go for reading at 340 nm.....Best regards-Deepti
Could you share with me the minimal concentration of total protein required to measure the malic enzyme activity? We are currently testing mitochondrial extract. We can see the protein (ME2) in western blot but no activity is detected!
Hi, I apologize but at the moment I am in the process of moving and I do not have much time to answer exactly on your question. Do you have a test enzyme activity in mitochondrial extract to be sure that enzymes are "alive" in the extract?
Chirstelle we are working on cytosolic Malic enzyme of Oleaginous yeast. This enzyme has specificity more for NADP+ rather than NAD+ . Alexander, thanks for your inputs. We have been able to troubleshoot the problem taking into consideration your inputs. Good luck Christelle for the research. I think Alexander rightly mentioned that though the enzyme may be present but there is a substantial loss in activity if the preservation of samples is not done in proper way. Even sometimes the freshly harvested cells give very high activity but older cells show reduced activity. We are preserving cell extracts in -80 degree Celsius.
Christelle Bahlawane
Hi, Dear Christelle
I,m going to measure the malic enzyme activity in cancer cell and compare it between tow sensitive and resistance cells.
can you share your protocol and procedures with me?
thanks.
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