I want to estimate lipid peroxidation product i.e. MDA from serum. Method includes addition of TCA in first step but after addition serum is getting cloted. Would you please help me to resolve this problem.
The 'clot' will be denatured protein from the serum - this is probably the reason your method use TCA (I presume you mean Trichloroacetic acid). This is the usual method for protein removal from serum for a variety of assays. Precipitate the clot by centrifugation and you should have an essentially protein free sample.
The 'clot' will be denatured protein from the serum - this is probably the reason your method use TCA (I presume you mean Trichloroacetic acid). This is the usual method for protein removal from serum for a variety of assays. Precipitate the clot by centrifugation and you should have an essentially protein free sample.
This precipitate denatured proteins is normal with this procedure. Add first serum followed by TCA and TBA and you must prepare incubation (about 10 min). Then centrifuge and ready.
This is mine procedure:
0.1 mL serum
+
2 mL H2O
+
1 mL 20% TCA
+
1 mL 7 % TBA
incubation in 100 oC for 10 min
the reaction stop by placing in ice-cold water
centrifuge - 3000 rpm or more for 10 min
my another doubt is that how can we calculate the conc of MDA after getting value of absorbance.
supernatant extinction read at 532 nm
In my study total thiobarbituric acid-reactive substances (TBARs) were expressed as MDA, using a molar extinction coefficient for MDA of 1.56 x 105 cm−1 M−1; results were expressed as nmol (TBARs)/ml.
okk so i need to substract value of my blank from my test value and need to divide this value by extinction co efficient.Am i right?
Yes Paul Jones is correct. Because of low pH, proteins get denature and gets precipitate. That is the only usual routine protocol people are using. Just centrifuge the content and continue the further process.
Thanks a lot everyone.Finally i got my result.It was possible only because of you all.
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