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Product Details
| Cat # +Size | K627-100 |
|---|---|
| Size | 100 assays |
| Detection Method | Absorbance (450 nm) |
| Species Reactivity | Mammalian |
| Applications | Kit detects 0.02 mM – 10 mM lactate in various samples. |
| Features & Benefits | • Simple procedure; takes ~40 minutes • Fast and convenient • Kit contains the necessary reagents for accurate measurement of Lactate |
| Kit Components | • Lactate Assay Buffer • Lactate Enzyme Mix • L(+)-Lactate Standard (100 mM) |
| Storage Conditions | -20°C |
| Shipping Conditions | Gel Pack |
| USAGE | For Research Use Only! Not For Use in Humans. |
Details
Lactate (CH₃CH(OH)COO-) plays important roles in many biological processes. Abnormally high concentrations of lactate have been related to disease states such as diabetes and lactic acidosis, etc. L(+)-Lactate is the major lactate stereoisomer formed in human intermediary metabolism and is present in blood. D(-)-Lactate is also present but only at about 1-5% of the concentration of L(+)-Lactate. In the Lactate Assay Kit, lactate is oxidized by lactate dehydrogenase to generate a product which interacts with a probe to produce a color (λmax = 450 nm). The kit detect L(+)-Lactate in biological samples such as in serum or plasma, cells, culture and fermentation media. There is no need for pretreatment or purification of samples. It detects 0.02 mM – 10 mM lactate in various samples.
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Can one just measure the increase in NADH (corresponding to increased lactate) by measuring absorbance at OD340? Doesnt this correspond directly to lactate levels? Why is it necessary to measure production of formazan at OD450 intead?
Yes, theoretically it can be done by monitoring the changes in NADH absorption at 340 nm.
Issues: Low sensitivity and high interference and requires expensive quartz microplates/cuvettes.
The current enzymatic assays like K627 are a better way to get more accurate measurements because the signal is more specific and magnified allowing better sensitivity and accuracy.
Issues: Low sensitivity and high interference and requires expensive quartz microplates/cuvettes.
The current enzymatic assays like K627 are a better way to get more accurate measurements because the signal is more specific and magnified allowing better sensitivity and accuracy.
How many cells should we have in the cell culture to get results that fit in the standard curve?
Typically 1-2 million cells is recommended. This can vary depending on the Lactate content in the cells and hence might need to be optimized.
Is deproteinizing necessary for this assay?
Yes, deproteinizing is definitely recommended for metabolically active tissues and cells to ensure Lactate in the sample is not used up by enzymes like LDH. Samples can be stored if needed at -80C after deproteinizing for running the assay later. For media, it is not as critical but still recommended for best results.
What is the difference between K607 and K627?
K627 provides an alternative wavelength, as many customers have the 450 nm wavelength filter available in comparison to the 570nm wavelength filter required for the colorimetric version of K607. For samples containing 0.02 mM-10 mM Lactate K627 can be used.
K607 can be both colorimetric and fluorometric.
K607 can be both colorimetric and fluorometric.
How much tissue should be used?
The amount of tissue depends on the Lactate content and the metabolic activity level of the tissue. 10-100mg tissue homogenized in 3-4 volumes of assay buffer can be centrifuged and the supernatant used for the assay. We recommend testing a few different volumes of the homogenate to ensure the readings are within the linear range fo the std. curve.
Can medium with phenol red and FBS be used for this assay?
Phenol red will be fine for this assay since small volume of the medium is used per well anyway and after adding assay buffer to fill up the volume, the color is insignificant. This kit measures at 450nm which is an added advantage. FBS containing medium should be deproteinized before use with the assay.
What components need to be avoided in the medium to assay lactate in cells only?
Ideally, the medium should be devoid of Lactate and pyruvate. Pyruvate can act as a source of lactate for the cells. If cells are grown in medium containing these, simply remove medium, wash with PBS and then lyse the cells to measure intracelllular lactate.
Are trial sizes of this kit available?
Unfortunately, we do not have trial sizes of this kit available. However,for customers based in the US or Canada, a 10% off list price introductory discount can be offered on the list price. For all other customers based out of this area our regional BioVision distributor should be contacted.
Can individual components of this kit be purchased separately?
Yes, any of the kits components can be purchased separately without having to buy the whole kit. Please refer to the component Cat #s mentioned on the datasheet for ordering.
Is it possible to 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.
Can this kit be used to measure lactate in bacterial cells/medium?
Although we have not tested this kit with bacteria, since Lactate is the same across kingdoms, this kit should work. Bacterial cells with cell walls might need special lysis reagents.
Could you please provide a sample preparation protocol for cells and tissues?
Homogenize 1 x 106 cell or 10 mg of tissue in 100 μl of lactate assay buffer provided with the kit. You can use either a dounce homogenizer or sonicate on ice to homogenize and lyse the cells. Centrifuge the lysate at 10,000 g for 10 min and take the supernatant for the assay. Supernatant is then filtered through a 10kDa MW spin filter (BioVision, Cat.# 1997-25) to remove all enzymes that may degrade lactate. Note: if you want to use more cells/tissues, increase the amount of assay buffer proportionally to make sure that the lysis is complete.
Ling, Z. et al., (2017) miR-361-5p modulates metabolism and autophagy via the Sp1-mediated regulation of PKM2 in prostate cancer, Oncology Reports, Jul.2017, 38(3), 1621-1628
Sun, X. et al., (2017) MicroRNA-143 suppresses oral squamous cell carcinoma cell growth, invasion and glucose metabolism through targeting hexokinase 2, Bioscience reports, Jun.2017, 37(3), BSR20160404
Elmorsy, E. et al. (2017) Adverse effects of anti-tuberculosis drugs on HepG2 cell bioenergetics Hum Exp Toxicol. 2017 Jun;36(6):616-625.
Sun, Lucia et al. (2017) MicroRNA-143 suppresses oral squamous cell carcinoma (OSCC) cell growth, invasion and glucose metabolism through targeting Hexokinase2, Biosci Rep. 2017 Jun 8;37(3).
Miyata, Saori et al. (2017) Antimicrobial photodynamic activity and cytocompatibility of Au25(Capt)18 clusters photoexcited by blue LED light irradiation, Int J Nanomedicine. 2017 Apr 4;12:2703-2716.
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