Protein oxidation inhibition
To 100 μl BSA (10 mg -ml in 10 mM phosphate buffer, pH7.4), 100 μl FeCl2 (4 mM in 10 mM phosphate buffer, pH 7.4), 100 μl H2O2/ABTS (15mM for H2O2 and 25mM for ABTS in 10mM phosphate buffer, pH 7.4) and 75μl millet extract was added and incubated for 1h at 370C. The reaction was terminated with the addition of 0.2 ml 1M mannitol-0.5M EDTA solution. From this reaction mixture, 20 μg of protein samples were subjected to 12.5 % SDS-PAGE and band intensity was estimated using the Scion Image Beta 4.02 software (Mayo et al., 2003). Further, to the reaction mixture described above, 0.5 ml 10 mM DNPH was added and incubated for 1h at 37 0C. Protein in the sample mixture was precipitated with the addition of 0.625 ml 50% ice cold trichloroacetic acid.
Can anyone please clarify me why we are using only 50% ice cold trichloroacetic acid in this method to precipitate protein?
Reference:
Mayo JC, Tan DX, Sainz RM, Natarajan M, Lopez-Burillo S, Reiter RJ (2003) Protection against oxidative protein damage induced by metal-catalyzed reaction or alkylperoxyl radicals: comparative effects of melatonin and other antioxidants. Biochimica et Biophysica Acta (BBA)-General Subjects 1620(1):139-150.
TCA is a strong acid used as a reagent to stop enzymatic reactions and precipitate any protein present, which can then be removed by centrifugation. Depending on the purpose of the experiment, either the precipitated protein pellet or the clear supernatant are used for further analysis.
Btw, if you want to use the protein pellet (say, for electrophoresis or for determination of protein concentration), but there is too little protein present to give a good pellet, you can use deoxycholate as carrier:
@ARTICLE{Ben-76,
title = {Assay of proteins in the presence of interfering materials},
author = {A. Bensadoun and D. Weinstein},
journal = {Anal. Biochem.},
year = {1976},
pages = {241-250},
volume = {70},
doi = {10.1016/S0003-2697(76)80064-4},
language = {engl},
}
Dear Sir,
I'm really thankful to you in the context of your kind reply. Sir, I just have one more doubt regarding the same. The concentration of Trichloroacetic acid is 50% which reduces to 18-20% when added in the reaction mixture. However the protein concentration in the solution is around 1mg. Based on previous literature i could understand that 4-6% of tricholoroacetic acid is sufficient to precipitate around 90% of the protein of 1mg/ml concentration. Moreover the efficiency of TCA to precipitate proteins decreases at higher concentrations. It may precipitate upto only 80-85% of protein at 18-20% TCA concentration. Hence, my question is, in this case why in the procedure they have mentioned 50% TCA, when 4-6% of final TCA concentration is only sufficient to precipitate protein.
Yours sincerely
Sakthi kumaran
Research scholar
CSIR-CFTRI
I think that in this method, the TCA precipitation is used to separate the protein from the excess reagents. The precipitated protein is then washed with organic solvents and redissolved in guanidine HCl. DNP derivatization of the protein is then measured by absorbance.
This is the reference for the method:
Levine R.L., Garland D., Oliver C.N., Amici A., Climent I., Lenz A.G., Ahn B.W., Shaltiel S., Stadtman E.R. Determination of carbonyl content in oxidatively modified proteins. Methods Enzymol. 1990;186:464–478
I have used 30% TCA to mix with my samples and to get 15% TCA final concentration to precipitated proteins. This concentration was enough to precipitated all the protein.
J Agric Food Chem. 2015 Nov 11;63(44):9820-30. doi: 10.1021/acs.jafc.5b03563. Epub 2015 Oct 29.
Impact of Lipid and Protein Co-oxidation on Digestibility of Dairy Proteins in Oil-in-Water (O/W) Emulsions.
Obando M1,2, Papastergiadis A1, Li S1, De Meulenaer B1.
@Monica Obando and @Adam B Shapiro
Is there any research article which describes the phenomenon that High concentration of TCA (around 15-18%) is required to precipitate protein, in presence of interfering compounds such as DNPH?
It's not about quite the same topic, but this article gives some interesting insights into TCA precipitation that might be of use to you.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2771300/
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