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Product Details
| Cat # +Size | K552-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (570 nm) or Fluorescence (Ex/Em 535/587 nm) |
| Species Reactivity | Mammalian |
| Applications | Aspartate can be quantified in the range between 0.1–10 nmoles/well (2-200 µM). |
| Features & Benefits | • Simple procedure; takes less than ~40 minutes • Fast and convenient |
| Kit Components | • Aspartate Assay Buffer • Probe (DMSO solution) • Serum CleanUp Mix • Aspartate Enzyme Mix • Conversion Mix • Aspartate Standard (100 mM) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
L-Aspartic acid (Asp) is one of the 20 proteinogenic amino acids as building block for protein. Although a non-essential amino acid in mammals, it is a precursor to several other amino acids including four essential amino acids (Met, Thr, Ile and Lys). Aspartate is a metabolite in the urea cycle, participates in gluconeogenesis and transports reducing equivalents between the cytosol and the mitochondria via the malate-aspartate shuttle. Aspartate also stimulates NMDA receptors (not as strongly as glutamate) and hence serves as an excitatory neurotransmitter in the brain and is an excitotoxin. BioVision’s Aspartate Assay Kit provides a simple, convenient assay to measure aspartate in a variety of samples. In the assay, aspartate is converted to pyruvate which is oxidized with the conversion of a probe into a highly colored (570 nm) and fluorescent (Ex/Em 535/587 nm) species proportional to the amount of aspartate in samples. Aspartate can be quantified in the range between 0.1–10 nmoles/well (2-200 µM).
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How many cells should I use for the assay? In which volume of Aspartate assay buffer should I resuspend them?
Start with ˜2x10^6 cells, suspend the cell pellet in 500 µl of the aspartate assay buffer on ice. Homogenize using a Dounce homogenizer/or sonicator on ice, until efficient lysis is confirmed by viewing the cells under the microscope. Spin down the sample and collect the supernatant. Use the eluate for subsequent assays.
Should I use the colorimetric or the fluorometric assay?
The fluorometric method is not more accurate, but at least 10 fold more sensitive than the colorimetric method. The fluorometric assay is recommended for serum samples due to the low levels of aspartate.
Approximately which dilution of my samples should I use?
Samples that are metabolically active might have high levels of pyruvate which can generate background in the assay and hence the background control becomes important. You will need to test and identify appropriate dilutions of the sample in order ensure the readings will fall within the linear range of the standard curve.
Can I sonicate the samples in cold instead of using a Dounce homogenizer?
Sonication can be used instead of Dounce homogenization, but Dounce homogenization is gentle yet very effective. You need to makes sure heat is not being generated during sonication by using shorter pulses and low intensity while keeping the samples on ice.
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.
Gaglio et al., Oncogenic K-Ras decouples glucose and glutamine metabolism to support cancer cell growth. Mol Syst Biol, Jul 2014; 7: 523.
For more citations of this product click here
