Frequently Asked Questions for BIOXYTECH®
MDA/ 4-Hydroxyalkenals LPO-586 Assay
| Catalog Number: 21012|
|Format page for printing|
|How long does the test take to perform?|
|Reagent Preparation: 2 minutes
Sample Preparation: 20 minutes
Assay Procedure: 50-65 minutes|
|What is the sensitivity of the LPO-586 method?|
|Assay curve 0 to 4|
|What samples can be tested for MDA?|
|EDTA plasma, tissue homogenates, or cell lysates. The amount of free MDA in normal plasma or serum is at or below the limit of detection of the LPO-586 assay. If one wishes to determine levels of MDA in plasma, the MDA-586 assays kit is designed to obtain this type of data. Please contact OXIS for details. |
|How are samples prepared prior to testing?|
If necessary, remove blood by perfusion in situ with isotonic saline or in vitro by rinsing with ice cold isotonic saline (i.e., 0.9% NaCI).
Prepare a 20%-30% tissue homogenate (2-3 g tissue in 10 ml, buffer) in 20 mM phosphate-buffered saline, pH 7.4 containing 5mM butylated hydroxytoluene. Other buffers may be used, but the researcher should confirm non-interference in the assay by measuring the MDA and/or 4-HNE standards diluted in the chosen buffer. All homogenizing buffers should contain 5mM butylated hydroxytoluene (BHT) to prevent sample oxidation.
Centrifuge the homogenate to remove large particles (e.g., 3000 X g at 4|
|What is the stability of the assay reaction?|
|1 hour at room temperature or 2 hours refrigerated if samples are stored in the dark and no evaporation occurs|
|Is a standard needed to perform the test?|
|Yes. A standard is provided in the kit.
1x1 mL 10 mM 1,1,3,3-tetramethoxypropane (MDA) standard |
|What is the shelf life of the LPO-586 assay kit?|
|1 year from date of manufacture when refrigerated.|
|How is MDA concentration calculated in the procedure?|
|Calculating the concentration of analyte in a sample:
A A586 of sample
Asb Sample blank
Ao Reagent blank
e Molar extinction coefficient at 586 nm = 10,000
Concentration of analyte = ([A586 - Asb - Am] x 5)/e
|What are the advantages of the LPO-586 method?|
|No high temperature acid hydrolysis is required.
Reagents are stable and ready to use.
Simple assay protocol. |
|Even though I follow the LPO-586 protocol the BHT precipitates out of the homogenization buffer. Will this be a problem when I run the assay?|
|The BHT is not soluble at the suggested concentration so it is normal to see the precipitate. The precipitate will not effect the assay however as it will spin out during the centrifugation/clarification step (protocol currently under revision).|
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