We tried to do those measurements with a commercial colorimetric assay kit, but finally found its sensitivity does not fit our case. That can only give accurate readings for 10pmol changes which is definitely too high for us.
Does anyone have already got a convenient and sensitive method to measure these redox co-factors in bacteria cells? The method should be able to tell the difference at 0.1pmol level or at most 1pmol level.
I noticed some method on some papers, such as the one I attached. But I didnot find how sensitivity that method can be.
Much appreciate if anyone can help and give some good advice.
Bin,
As you have already discovered, colorimetric methods have their limitations at the low end. Methods that employ chemiluminescence can go an order of magnitude lower.
There are methods based on capillary electrophoresis where a single cell can be lysed, NAD+ and NADH separated and measured - sensitivity for NAD+ reaches well below 1 attomole.
Hi Bin,
Go through the following thread.
https://www.researchgate.net/post/What_is_the_best_way_to_measure_levels_of_NAD_NADH_in_frozen_tissue_samples
Dear Bin Lai
I would use live bacteria auto fluorescence (exc 340 emission 470nm) or even absorption at 340nm mostly NADH derived. To get the bacterial autofluorescence deplete them from NADH after to have the control. You might be able to use the Hoechst filter set on a fluorescence microscope with ccd camera, better EMCCD or scientific cmos) as well better if you have a bandpass emission filter around 450-500nm, microscopy/imaging guys in your lab might know.
regards
Heiko
Dear Heiko Dussmann,
Thanks. I also find some commercial cell-based assay kits to do that, and their standard curve is based on the cell numbers and fluorescence density. In my case, it will be hard to do this as I am culturing the cells under different conditions but not only different ambient conditions.
But the method you mentioned is quite interesting, may I ask more details?
1. how to do the control? I mean a good control should have the same metabolic activity of the sample, but this seems to be hard to be controlled. Taking an average of triple or experiments?
2. how to do the base line and standard curve? if we want to quantify the concentration, it will be necessary to have these information. or this method cannot do quantification?
3. Is that this method is developed for comparing different engineered strain or mutants?
Thanks.
the benefit of the auto fluorescence measurement is that you do not have to rely on an enzymatic activity in an assay. But you will have a relatively low signal above your auto fluorescence background.
1. control: 3 experiments under control conditions e.g. no mutant/ no drugs, check the bacterial autofluorescence by depleting them from NADH e.g. treat them with a non fluorescent compound to kill them, use an ionophor (e.g. 1uM FCCP), etc.
2. Quantification is done with a calibration curve derived from NADH in different concentrations in solution at same pH and temperature.
3. It is a relatively old method but was used in a lot of different systems. We used this method to detect relative changes over time using time lapse live cell imaging.
Recently there was also a publication about a FRET based NADH sensor called PEREDOX which you could get via addgene. I do not know if that one is available for bacterial expression since we were are currently testing it in cells, just have a look.
Hi, Heiko,
Thanks for your detailed response. It may not be suitable for my case but it is really good to know this method. To test the living cells, I also just got a response from a colleague who is experienced in testing these co-factors, and he gives a method based on biosensor, session 2.(b) in the paper I attached. Hope this may also interest you as well.
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To those who interest in this topic, a supplemental note here is it need to quite care about the extraction. To kill the enzyme activity, a quenching process is necessary. Quenching can be done by fast filtration or more commonly, the cold methanol. For both the method, it will have serious leakage during quenching, especially for methanol method. The leakage for fast filtration method can be somewhat overcome by washed by culture medium, not water; but for the cold method method, it is hard to avoid leakage, and 80% of the leakage happened in seconds. So, it has been a common mistake for researchers who use this method to exact the metabolites from centrifuged cells but throw the supernatant away. Actually, the supernatant contains more metabolites than the cells, especially for gram-negative cells.
More details can be found in the followed paper.
Article Sampling for Metabolome Analysis of Microorganisms
Hi, Lumeng,
I couldnot find any more sensitive assay kit to do the measurement, and therefore, the method I adapted is to concentrate my samples. I used cold methanol to extract the co-factors, and then freeze-dry the extractant to get lyophilized powder. Then you can resuspend the powder in small volume of buffer to get a reasonable concentrated stock solution for your assay kit. You need to do a preliminary test to determine how much cells you need for the extraction, and how much buffer used to resuspend the powder. The good way to start with is using as much cells as you can.
Good luck.
Bin,
As you have already discovered, colorimetric methods have their limitations at the low end. Methods that employ chemiluminescence can go an order of magnitude lower.
There are methods based on capillary electrophoresis where a single cell can be lysed, NAD+ and NADH separated and measured - sensitivity for NAD+ reaches well below 1 attomole.
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