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Lipid Peroxidation (MDA) Colorimetric/Fluorometric Assay Kit

based on 5 citations in multiple journalsLipid Peroxidation (MDA) Colorimetric/Fluorometric Assay Kit54.1 4
Catalog #: K739
$455.00

Product Details

Cat # +Size K739-100
Size 100 assays
Detection Method Absorbance (532 nm) or Fluorescence (Ex/Em 532/553 nm)
Species Reactivity All
Applications The kit detects MDA levels as low as 1 nmol/well colorimetrically and 0.1 nmol/well fluorometrically.
Features & Benefits • Simple procedure
• Fast and convenient
• Sensitive assay for measuring lipid peroxidation (LPA) in a wide range of samples
Kit Components • MDA Lysis Buffer
• Phosphotungstic Acid Solution
• BHT (100X)
• TBA
• MDA Standard (4.17 M)
Storage Conditions -20°C
Shipping Conditions Gel Pack
USAGE For Research Use Only! Not For Use in Humans.

Details

Quantification of lipid peroxidation is essential to assess oxidative stress in pathophysiological processes. Lipid peroxidation forms Malondialdehyde (MDA) and 4-hydroxynonenal (4-HNE), as natural bi-products. Measuring the end products of lipid peroxidation is one of the most widely accepted assays for oxidative damage. BioVision’s Lipid Peroxidation Assay Kit provides a convenient tool for sensitive detection of the MDA in a variety of samples. The MDA in the sample is reacted with Thiobarbituric Acid (TBA) to generate the MDA-TBA adduct. The MDA-TBA adduct can be easily quantified colorimetrically (λ = 532 nm) or fluorometrically (Ex/Em = 532/553 nm). This assay detects MDA levels as low as 1 nmol/well colorimetrically and 0.1 nmol/well fluorometrically.


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Please recommend the steps to fully dissolve the TBA solution if there is precipiate after bringing up the volume to 25ml with water?
Sonication in a RT water bath can be used if needed. You can also dissolve the TBA in 15 ml of 50% glacial acetic acid in water. Then fill the volume with water to make 25ml. Mix well. This should help in dissolving the TBA. If there is still little precipitate, use 600 ul as directed and any particle in suspension will dissolve at 95C when incubated for 60 mins.
Can urine samples be used with this kit?
Urine samples can be used directly with this kit. The samples should be assayed immediately after collection for best results. If the assay is to be performed later, the samples need to be stored at -70C.
Can tissue frozen in liquid nitrogen be used in this assay?
Although not ideal, flash frozen tissue samples can be used to prepare homogenates and then the lysates can be used with the kit. Once thawed, follow the instructions on our datasheet for preparing the lysate. Process the samples quickly on ice to prevent any loss. Bear in mind that the results obtained with fresh and frozen samples can differ from each other.
What is the lower detection limit for this assay?
The assay can measure down to 1 nmol per well colorimetrically and 0.1 nmol per well fluorometrically.
When resuspending the pellet with the water/BHT solution, it does not dissolve. Is there a way to better resuspend? What about after the assay reaction, if there is a precipitate in the TBA solution after the 95C incubation, is there loss of MDA there, or will it all be in the MDA-TBA adduct in the solution?
If you have difficulty resuspending the pellet in the 100 µl ddH2O (with 2 µl BHT), after the final volume adjust to 200 µl with ddH2O, the pellet should be solubilized. You can use mild water bath sonication to dissolve the pellet once you have pipetted up and down to break up the initial pellet.If there is turbidity or precipitate formation after heating with TBA, filter the sample as mentioned on the datasheet.
Why do we use the pellet from plasma samples but the supernatant from tissue samples?
For tissues, PCA precipitates all proteins and the lipids are in the supernatant. For plasma samples, phosphotungtic acid precipitates lipids and hence the pellet is used.
Under what situation centrifugation after homogenizing the sample with 300 ul of MDA lysis buffer is sufficient compared to the sample being homogenized in water and then the protein is precipitated by perchloric acid. Is there any major difference between these two methods for preparing the sample?
The lysis buffer will help disintegrate tissue and lyse cells and since it has SDS, it will denature proteins. Then, once homogenized, the lysate can be centrifuged (debris, nuclei and proteins precipitate). If the tissue is difficult to homogenize in water, I would use this method. Adding PCA will precipitate proteins in the alternative method. Either could work equally well. Effective homogenization is key.
Does mild hemolysis of the plasma affect this assay?
Acid precipitation will remove all proteins including hemoglobin. Moreover, after the addition of PTA solution and centrifugation, the pellet is collected and dissolved in water before being used for the assay. The amount of sample added per well should retain none or insignificant amounts of hemoglobin and this should not affect the readings. Also, the fluorometric method is not affected by hemolysis.
Does this kit detect free MDA, bound-MDA or total MDA?
Effectively, we detect MDA which is free to form an adduct with TBA. If MDA in the sample is bound to collagen or other proteins, this will not be detected unless released. The acid treatment precipitates all proteins, so we expect the MDA in the sample to be free and hence total MDA can be detected.
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.
Can alternate buffers be used 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.
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.
Would you mind telling us what the color of the assay product, the MDA-TBA adduct, in K739 is? We found the color of the Sample MDA-TBA adduct to be green and pink for Standards? Would you please advise whether this is normal?
The Standards will yield a pink color as it is pure MDA and when it reacts with TBA, you can expect a pinkish product. Because Samples are not pure MDA, the miscellaneous compounds in the Sample sometimes react with TBA, giving a color, which generally does not interfere with the quantification of the TBA-MDA adduct.
Badreldin H. Ali, Testicular Toxicity of Water Pipe Smoke Exposure in Mice and the Effect of Treatment with Nootkatone Thereon. Oxid Med Cell Longev, June 2019;  31341528.
Sarah H. Merkling, Peroxisome-associated Sgroppino links fat metabolism with survival after RNA virus infection in Drosophila. Sci Rep., Feb 2019;  30765784.
Mostafa Ibrahim Waly, The Protective Effect of Curcumin against Nitrosamine-Induced Gastric Oxidative Stress in Rats. Prev Nutr Food Sci, Dec 2018; 30675457.
Marni E. Cueno, et al., (2018) Gingival Periodontal Disease (PD) Level-Butyric Acid Affects the Systemic Blood and Brain Organ: Insights Into the Systemic Inflammation of Periodontal Disease, Frontiers in Microbiology, Jun. 2018, 29915575
Yanru Zhao, et al., (2018) MiR-124 aggravates failing hearts by suppressing CD151-facilitated angiogenesis in heart, Oncotarget, Jan. 2018, 29581851
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