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PEP Colorimetric/Fluorometric Assay Kit

Catalog #: K365

Product Details

Cat # +Size K365-100
Size 100 assays
Detection Method Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm)
Species Reactivity Mammalian
Applications The detection limit is approximately 1 µM PEP in biological samples.
Features & Benefits • Simple procedure
• Fast and convenient
• The assay is sensitive, stable and high-throughput adaptable.
Kit Components • PEP Assay Buffer
• PEP Probe (in DMSO)
• PEP Converter
• PEP Developer Mix
• PEP Standard (1 μmole)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.


Phosphoenolpyruvate (PEP) is an important intermediate in carbohydrate metabolism. Containing a high-energy phosphate bond, PEP is involved in glycolysis and gluco-neogenesis and in the shikimate pathway as well as in carbon fixation in plants. Bacteria utilize PEP in the phosphor-transferase system to acquire sugars from the environment. In the glycolytic pathway, PEP is formed from 2-phosphoglycerate by enolase and generates ATP through the action of pyruvate kinase. BioVision’s PEP Assay Kit provides a convenient colorimetric and fluorometric means to measure PEP levels in various samples. In the assay, PEP is converted to ATP and pyruvate. The generated pyruvate is quantified by colorimetric (λmax = 570 nm) or fluorometric methods (Ex/Em = 535/587 nm). The assay is simple, sensitive and reliable. The detection limit is approximately 1 µM PEP in biological samples.

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We received an inquiry from one of our customers in regard to the following product: #K365-100, PEP Colorimetric/Fluorometric Assay Kit. The customer would like to know the way to prepare cell samples. Is it similar to the way to prepare tissue samples described in the datasheet?
Yes, please use the same protocol as for tissues: Cells (2-5 x 10^6) should be homogenized in 100 µl ice cold HClO4 and vortexed until the contents are thoroughly mixed. Neutralize carefully by adding repeated small aliquots (~10 µl per aliquot) of KHCO3 followed by vortexing. Final pH should be 6.5-7.5. Centrifuge at 12,000g for 3 minutes. Some plant extracts need to be decolorized with activated charcoal (5 mg per tube) which can be added and vortexed prior to the centrifugation step. Use samples immediately or store at -80°C. Add up to 50 µl sample per well in a 96-well plate; bring the volume to 50 µl with Assay Buffer.
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.
Emily D. Montal, Inhibition of phosphoenolpyruvate carboxykinase blocks lactate utilization and impairs tumor growth in colorectal cancer. Cancer Metab., Aug 2019; 31388420.
For more citations of this product click here