May be problem with a kit

I'm so excited that find someone have the same problem with me which confused me a lot and still don't know the reason. I have purchased the kit from Biovision and Sigma (Cat MAK039) respectively for Acetyl-Coenzyme A detection. Both kits' results showed higher signal in blank (without converting enzyme) than corresponding sample (the same extract with converting enzyme).

In addition, I have used the α-Ketoglutarate Assat Kit (Cat MAK054) from sigma which utilize the similar principle that transform α-KG to pyruvate catalyzed by the 'converting enzyme' , and the pyruvate was detected in colorimetric or fluorometric after a coupled enzyme assay . The same abnormal results showed that the blank which should represented the pyruvate in the background was higher than group with converting enzyme.

My samples were extracted from cancer cells (about 1x106 cells for a sample)by assay buffer or PBS using sonicate thoroughly. And I have done the deproteinze procedure using 10 kDa MWCO spin filter to avoid the interference of the enzymes.

I don't know how to fix the problem and if those kits were really available for detection in cells or still missed some matters need attention.

Dear Bingluo,

after a lot of thinking and reading papers and manuals couple months ago, I realized that the problem might be in oxidized (disulfide) form of CoA. The protocol includes the step of "masking" free CoA (I'm 99% sure it utilizes N-ethyl maleimide, but could be pyridine derivatives) and then removing the excess "masking agent" (by using glutathione (99% sure) or other SH-containing reagents). However, what glutathione does is not just reacting with excess N-ethylmaleimide, but reducing CoA disulfide, whose level, depending on your sample preparation and storage, can be musch higher, than those of Acetyl-CoA. The equilibrium in CoASSCoA + 2GSH = 2 CoASH + GSSG is to the left, but since CoA further reacts in indicator reaction, CoA disulfide is being slowly reduced and signal is slowly accumulating.

As a result, with or without "converting enzyme", the amount of CoA is enough to get a signal by using coupled enzyme (99% sure it's 2-oxoglutarate dehydrogenase complex and synthetic dye (PicoProbe; could be resazurin or its derivative) which is transformed to light-absorbing/fluorescent compound by electron transfer from NADH).

"Converting enzyme" may contain something that absorbs light at emission wavelength of PicoProbe, resulting in lower fluorescence signal than blank without the "enzyme".

Now I believe the kit won't work anyway with my brain extracts.

But couple weeks ago I decided to try again with other methods, for example using home-made coupled enzymatic system including purified malate dehydrogenase and citrate synthase, for which neither CoA, nor its disulfide should interfere. There was some signal, but too weak for accurate quantitative determination.

Reducing of all disulfides in extracts with dithiothreitol and then adding high excess of N-ethylmaleimide, followed by even higher excess of glutathione, also didn't work.

Now I am waiting for additional enzyme (phosphotransacetylase), which could increase the senitivity of the assay.

After it arrives to the lab, I'll try again if there is time.

Can't help with a-KG assay kit, but the system you described might include transamination reaction catalyzed by alanine aminotransferase, coupled reaction catalyzed by lactate dehydrogenase, followed by reduction of the same synthetic dye. Due to the interference from endogenous puruvate (whose level may be higher than a-KG), I'd suggest using home-made assay system with purified 2-oxoglutarate dehydrogenase complex (look at Sigma-Aldrich) and its substrates (NAD+, CoA) and coenzymes (thiamine pyrophosphate, MgCl2, CaCl2, dithiothreitol) and detection of NADH absorbance at 340 nm. CoA may be expensive though.

Thank you very much for your careful and professional sharing. When I have time and conditions, I will re-improve and try and communicate with you.

It is known that Free CoASH, malonyl CoA, and succ-CoA in samples generate background. In order to correct for this background, add 10 µl of CoA Quencher to each Standard, Sample and background sample to quench free CoA. This is mentioned in step 2 under section V. Acetyl CoA Assay Protocol. Did you add the CoA Quencher to quench free CoA?

Dear Shyamali, sorry for such an adjourned response. Yes, I do the assay exactly as described in protocol. The problem seems to be in free CoA which is oxidized and thus, not quenched by "Quencher" but reduced back by "Quench remover".

Update: the kit actually worked with liver extracts, but only prepared the same day as the day of assay. When stored even for a week at -70 degC, the extracts give too much of background to measure the acetyl-CoA level (with and without the "converting enzyme" the signals are the same), indicating the rapid free CoA oxidation during storage interferes with the assay.

For brain tissue the amount of acetyl-CoA seems too low to determine with the kit, doesn't matter is the extract freshly prepared or stored.

Therefore, the kit is probably ok, but can be used for limited number of objects.

I got the Acetyl CoA assay from Sigma and I had exaclty the same problem.

I'm trying to measure Acety CoA from macrophages, and my background is around 30.000 RFU, while my samples are around 22.000.

Artem V. Artiukhov do you have any idea of how I could reduce this oxidation of free CoA?

I did the assay with fresh samples following protocol available on sigma website.

Thank you a lot!

Dear Marina Gomes Machado ,

I believe the oxidation of free CoA occurs at the moment of cell lysis and not much can be done about it. There are some antioxidants that can be added (like dithiothreitol), but most will affect masking stage of the non-oxidized CoA, so it won't work anyway with these kits.

At the moment we've switched to measurement of other metabolic indicators, but acetyl-CoA still can be measured, e.g. via mass-spectrometry, if there is a necessary equipment and qualified personnel near you...