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PicoProbe™Acetyl-CoA Fluorometric Assay Kit

based on 11 citations in multiple journalsPicoProbe™Acetyl-CoA Fluorometric Assay Kit115 5
Highly sensitive assay
Catalog #: K317
$450.00

Product Details

Cat # +Size K317-100
Size 100 assays
Detection Method Fluorescence (Ex/Em 535/587 nm)
Species Reactivity Mammalian
Applications The assay can detect 10 to 1000 pmol of Acetyl CoA (with detection limit ~0.4 μM) in a variety of samples.
Features & Benefits • Very simple procedure
• Convenient
• BioVision has developed a high sensitivity assay for Acetyl CoA level in a variety of biological samples. In the assay, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with PicoProbe to generate fluorescence.
Kit Components • Acetyl CoA Assay Buffer
• PicoProbe
• Conversion Enzyme
• Acetyl CoA Enzyme Mix
• Acetyl CoA Substrate Mix
• CoA Quencher
• Quench Remover
• Acetyl CoA Standard (10 µmol)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Acetyl CoA is a central molecule of metabolism. It carries acetate, used in the build-up and breakdown of larger molecules. Acetyl CoA is key in synthetic pathways leading to sesquiterpenes, precursors to cholesterol and other sterols, flavenoids and other polyketides, polyenes and long-chain fatty acids. It is the source of the acetyl group used in histone acetylation. The acetyl group is also incorporated into a variety of other molecules such as acetylcholine, melatonin, heme and TCA cycle intermediates. BioVision has developed a highly sensitive assay for determining Acetyl CoA level in a variety of biological samples. In the assay, free CoA is quenched then Acetyl CoA is converted to CoA. The CoA is reacted to form NADH which interacts with PicoProbe to generate fluorescence (Ex=535/Em=587 nm). The assay can detect 10 to 1000 pmol of Acetyl CoA (with detection limit ~0.4 µM) in a variety of samples.


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We have received an enquiry regarding PicoProbe™ Acetyl CoA Assay Kit, product code K317-100. I would appreciate if you are able to provide a protocol for preparing cell culture samples ready for this assay.
For preparation of cell culture samples follow this protocol: Cell Lysis Buffer (1X):
  • 20 mM Tris (pH 7.5)
  • 150 mM NaCl
  • 1 mM EDTA
  • 1 mM EGTA
  • 1% Triton X-100
  • 2.5 mM sodium pyrophosphate
  • 1 mM ?-Glycerolphosphate
  • 1 mM Na3VO4
  • 1 µg/ml Leupeptin
Note: We recommend adding 1 mM PMSF before use.
Preparing Cell Lysates
  • Aspirate media. Treat cells by adding fresh media containing regulator for desired time.
  • To harvest cells under nondenaturing conditions, remove media and rinse cells once with ice-cold PBS.
  • Remove PBS and add 0.5 ml 1X ice-cold Cell Lysis Buffer plus 1 mM PMSF to each plate (10 cm2) and incubate the plate on ice for 5 minutes.
  • Scrape cells off the plate and transfer to microcentrifuge tubes. Keep on ice.
  • Sonicate 4 times for 5 seconds each on ice.
  • Microcentrifuge for 10 minutes at 4°C, and transfer the supernatant to a new tube. The supernatant is the cell lysate.If necessary, lysate can be stored at –80°C.Once the lysate is ready, follow the deproteinization protocol as mentioned on the attached datasheet and then proceed with the whole protocol.
Could you please inform us how to prepare liver tissue samples for this assay?
Here is the protocol for tissue sample prep:Tissue samples: (20 - 1000 mg) should be frozen rapidly (liquid N2 or methanol/dry ice), weighed and pulverized. Add 2 µl 1N perchloric acid/mg sample. KEEP COLD! Homogenize or sonicate thoroughly. Spin homogenate at 10,000 x g. Neutralize supernatant with 3 M KHCO3, adding repeated 1 µl aliquots/10 µl supernatant while vortexing until bubble evolution ceases (2 - 5 aliquots). Put on ice for 5 min. Check pH (using 1 µl) should be ~ pH 6 - 8. Spin 2 min to pellet KClO4. Add 10 µl of sample into duplicate wells (Sample and Background) of a 96-well plate; bring volume to 50 µl with Assay Buffer.
I would like to assay with colorimetric plate reader and I could not find about which wavelength is suitable for it. If I want to do with colorimetric plate reader, should I modify assay protocol somehow?
This is a fluorometric assay. You cannot use a colorimetric plate reader with it.
When you add the CoA conversion to the samples which reaction mix should you add? The 0-1 nmol mix or the 0-100 pmol mix? Do you have to run your samples in duplicates and add 0-1 nmol mix to one and 1-100 pmol mix to the other.
Add the same range reaction mix as the standard you are making. For eg, if you suspect your samples to have low A CoA, use the 0-100 pmol standards and reaction mix.
First of all, for sample preparation from cultured cells, is it absolutely necessary to follow the specific cell lysis buffer provided in your protocol? Since we do have a particular cell lysis protocol we follow regularly, it would be nice to keep doing what we have done.Second, for the deproteinization step, you recommend the use of perchloric acid method, could this be substituted by using a 10kD MW cut-off spin column?
Yes it is very crucial to do the sample prep exactly as recommended as Acetyl CoA is very sensitive to degradation and you risk the chance of inefficient results if you make changes to the protocol. The deproteinization approach though not as sensitive as the sample prep, would still benefit from using PCA rather than the 10 kDa spin filters. But you can use the spin filters if you do not have the option of using PCA.
I simply want to follow the protocol in your instruction manual (K317-100) I find the manual information: 10 umol/vial, where100 ul of water have to be added. If I add 100 ul to 10 umol, I get 10 umol/100 ul (or 100nmol/ul) This results according to me, in a concentration of 100 mM (or 100 mmol/Liter). In your manual, the description leads to 10mM. Since initial concentrations are important for the standard curve, I would like to know (if possible step by step) how you get the concentration of 10 mM (as in your protocol) after adding 100 ul to 10 umol containing vial.
We provide you with a 1 µmol (not 1 µM) standard and ask you to reconstitute it with 100 µl water. That is 1 µmol or 1000 nmol in 100 µl of water, which would make the final amount of the standard to 10 nmol/µl as mentioned in the datasheet. The molarity or the conc of this 10 nmol/µl would be 10 mM. I think you are mistaking between the molar amount and the molar concentration or are wrongly assuming that the initial standard amount is 10 µmol, when is actually is only 1 µmol.
The de-proteinization protocol mentions KOH, then switches to KHCO3. Which is correct?
Both KOH and KHCO3 will perform the same neutralizing action. Either of them can be used with this kit.
Can this kit be used with samples like bacteria, plants, drosophila, yeast etc?
We have optimized the kit with mammalian samples. However, theoretically these kits should work with samples from multiple species/sources. Since the optimal conditions depend on the sample type, the protocol has to be be adapted to fit the samples for efficient results. Please refer to this kit's citations to see what kind of samples have been used with this kit other than mammalian samples.
Can we use frozen samples with this assay?
Fresh samples are always preferred over frozen samples. However, frozen samples can also be used, provided, they were frozen right after isolation, were not freeze thawed multiple time (for which we recommend aliquoting the samples before freezing) and have been frozen for relatively short periods.
Can we use a different wavelength than recommended for the final analysis?
It is always recommended to use the exact recommended wavelength for the most efficient results. However, most plate readers have flexibility in their band width of detection in increments of +/- 10 nm. Depending on this flexibility range, you can deviate from the recommended wavelengths within limits.
What is the exact volume of sample required for this assay?
There is no specific volume we can recommend for the amount any sample to be used since it is completely sample concentration and quality based. You have to do a pilot expt with multiple sample volumes to determine the optimal volume which gives a reading within the linear range of the standard curve. Please refer to the citations for this product to see what other clients have used with similar sample types.
Do you have trial sizes of this kit?
Unfortunately, we do not have trial sizes of this kit available. However, if you are based in the US or Canada, we will give you a 10% off list price introductory discount on its purchase price. If you are based out of this area please contact your regional BioVision distributor.
What is the shelf life of this kit?
This kit is good for 12 months from the date of shipment in the unopened form when stored at the appropriate temperature and appropriate conditions. After opening and reconstitution, some of the components in this kit are good for 2 months at -20°C. Please refer to the datasheet for storage information and shelf life of each of the components.
Why are my standard curve values lower than those shown on the datasheet?
There are multiple factors which influence the signals like the incubation times, room temperature, handling etc. In general, to increase the value of the standards, you can increase the incubation time. As long as the standard curve is linear, it should be fine to use, since all of your samples will also be measured under the same conditions on this curve.
How do I normalize my samples against protein concentration
You can use a protein quantitation assay on the supernatants you get from cell/tissue lysates or with any other liquid sample in the assay buffer.
Can we purchase individual components of this kit?
Yes, you can purchase any of the kit's components without the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Can we use an alternate buffer for sample preparation (cell lysis, sample dilutions etc)?
Our assay buffers are optimized for the reactions they are designed for. They not only contain some detergents for efficient lysis of your cells/tissue, but also contain some proprietary components required for the further reactions. Therefore, we highly recommend using the buffers provided in the kit for the best results.
Should I make a standard curve for every expt I do, or is one curve/kit enough?
Yes, I would strongly recommend you to do the standards every time you do the expt. There is always a chance that something was done differently that day and we do not want any conditions to differ between standards and samples.
It seems that the kit will convert the acetyl CoA into CoA which is then converted to form NADH, which will in turn interact with the PicoProbe. Did you test if, in the first step or one of the following conversions, there is any cross-reaction or interference from Aceto Acetyl-CoA?
Yes, we have tested the cross-reactivity of Aceto Acetyl-CoA and other CoA such as malonyl CoA, Succinyl CoA etc, which if present in your sample may generate some background. Therefore, we recommend doing a sample background control for your sample type. Please subtract background RFU from sample RFU before calculating the amount of Acetyl CoA in your samples.
Yan Ping Bai, Nicorandil alleviated cardiac hypoxia/reoxygenation-induced cytotoxicity via upregulating ketone body metabolism and ACAT1 activity. Korean J Physiol Pharmacol, Dec 2018;  30627008.
Kajimoto et al., Differential effects of octanoate and heptanoate on myocardial metabolism during extracorporeal membrane oxygenation in an infant swine model. Am J Physiol Heart Circ Physiol, Oct 2015; 309: H1157 - H1165.
Ehara et al., Ligand-Activated PPARα-Dependent DNA Demethylation Regulates the Fatty Acid β-Oxidation Genes in the Postnatal Liver. Diabetes, Mar 2015; 64: 775 - 784.
Kanda et al., ATP6AP2/(Pro)renin Receptor Contributes to Glucose Metabolism via Stabilizing the Pyruvate Dehydrogenase E1 β Subunit. J. Biol. Chem., Apr 2015; 290: 9690 - 9700.
Jung et al., Improvement of 2,3-Butanediol Yield in Klebsiella pneumoniae by Deletion of the Pyruvate Formate-Lyase Gene. Appl. Envir. Microbiol., Oct 2014; 80: 6195 - 6203.
For more citations of this product click here