• Detection method- Absorbance (450 nm)
• Sample type- Cell and Tissue culture supernatants, urine, plasma and serum, as well as many other biological fluids etc.
• Species reactivity- Mammalian
• Application- The kit can detect 1 -100 mU/ml of LDH directly in samples.
Features & Benefits:
• Simple procedure; takes ~ 30 minutes
• Fast and convenient
• Sensitive assays for measuring LDH activity in various biological samples and the kit is also suitable for high throughput analyses.
• LDH Assay Buffer
• LDH Substrate Mix (lyophilized)
• NADH Standard (0.5 µmol; lyophilized)
• LDH Positive Control
Lactate dehydrogenase (LDH) is an oxidoreductase (EC 22.214.171.124) present in a wide variety of organisms. It catalyses the interconversion of pyruvate and lactate with concomitant interconversion of NADH and NADᶧ. When disease or injury or toxic material damages tissues, cells release LDH into the bloodstream. Since LDH is a fairly stable enzyme, LDH has been widely used to evaluate the presence of damage and toxicity of tissue and cells. Quantification of LDH has broad range of applications. In this colorimetric LDH quantification assay, LDH reduces NAD to NADH, which then interacts with a specific probe to produce a color (λmax = 450 nm). The kit quantifies LDH activity in variety of biological samples such as in serum or plasma, cells, culture medium and fermentation, etc. The assay is quick, convenient, and sensitive. The kit can detect 1 -100 mU/ml of LDH directly in samples.
USAGE: For Research Use Only! Not For Use in Humans.
What is the detection range for this kit?
The detection range is 1 -100 mU/ml of LDH
Can this kit measure both intra- and extracellular LDH activity?
Yes, this kit can measure both intra and extra-cellular Pyruvate. For intracellular pyruvate measurement from cell samples: Start with ~1-2 million cells, suspend the cell pellet in 2-4 volumes of the pyruvate assay buffer on ice, pass it through the Dounce homogenizer (10-50 times) on ice, till efficient lysis is confirmed, by viewing the cells under the microscope. Spin down the sample, take the supernatant and use this supernatant for your subsequent assays. Appropriate dilutions/volume of the sample must be tested in order ensure the readings will fall within the linear range of the standard curve. For extracellular media, remove any cell debris by spinning down and optimize the volume needed to get values within the linear range of the std. curve.
How specific is this kit? It seems that this kit is only measuring the change between NAD and NADP, which can be catalyzed by enzymes other than LDH. Is there any way to be sure that the kit is measuring only LDH activity?
The principle of the assay is :LDH converts Lactate to pyruvate, generating NADH from NAD. The NADH interacts with the probe to generate color at 450nm. The amount of colored product formed is directly proportional to the LDH activity in the sample. The buffer conditions (pH, presence of an electron transfer agent) are optimized for LDH activity and since the NADH generation is coupled to Lactate conversion to pyruvate, this assay specifically detects LDH and not other NAD to NADH converting enzymes in the cell. Any nonspecific endogenous NADH can be accounted for by omitting the substrate int he reaction mix. This background value can be subtracted from the sample LDH activity value.
If I am measuring LDH alpha (LDH5), which converts pyruvate to lactate, will this assay work for me?
Lactate dehydrogenase catalyzes the interconversion of pyruvate and lactate with concomitant inter-conversion of NADH and NAD+. It converts pyruvate, the final product of glycolysis, to lactate when oxygen is absent or in short supply, and it performs the reverse reaction during the Cori cycle in the liver. So your LDH5 converts pyruvate to lactate and vice versa. In our assay, NAD (produced during pyruvate to lactate conversion by LDH5) is reduced to NADH, which interacts with a probe to produce a color (λmax = 450 nm). In essence, this assay can work for your objective. The reaction conditions and the relative amount of pyruvate and lactate decide which direction the reaction will go.
Does phenol red in the edia affect this assay?
Since 2-50 ul sample is added per well, the color from phenol red is diluted by the assay buffer/reaction mix. Typically this does not affect the assay. The OD 450 readings are in the yellow
How can adherent cells be used for this assay?
Trypsinization can be used to detach cells. Then collect the cells, spin down, remove medium+trypsin and then proceed with homogenization using the assay buffer in the kit. It is important to be careful since over-trypsinization might damage the plasma membrane and leak LDH into the medium.
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 essential to make a standard curve for every expt, or is one curve/kit enough?
Yes, it is strongly recommended 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.
Can frozen samples be used 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.