I am analyzing clam tissues for lipid peroxidation using the TBARS protocol. To extract lipids for MDA analysis, is it necessary to use PMSF when I am not interested in protein analysis?
This is a protease inhibitor. If there are proteases in the sample that can affect your results, this might be helpful. Quickest way to find out is to run an assay with/without PMSF and see if there's a difference.
Edith Etor
Which protocol are you following for estimating LPO activity in clam tissues? NOTE:- PMSF is not mandatory for every LPO protocol
Edith Etor
Not sure which article relates to Popovic et al. 2013 (Google has many articles of same author name - Popovic et al. 2013). I can only suggest if you send the article or give the link.
Anyhow, since you are quantifying LPO in clams, I suggest Niehaus & Samuelsson OR Ohkawa method (Both PDF's attached). These two do not use PMSF
PS - Once again about Popovich et al 2013 - Till I read the article, I cannot suggest the utility of PMSF for LPO method.
Proteek Dasgupta Thanks.
Here it is: Popović N, Beer Ljubić B, Strunjak-
Perović I, Babić S, Lorencin V, Jadan M, et al.
(2020) Seasonal antioxidant and biochemical
properties of the Northern Adriatic Pecten
jacobaeus. PLoS ONE 15(3): e0230539. https://doi.
org/10.1371/journal.pone.0230539
Proteek Dasgupta Thanks for the articles. I have read through them. I like the protocol used by Ohkawa for homogenising the tissues. My concern is using chloroform for extraction. I understand it has safety concerns. Some papers propose hexane-isopropanol but this has been reported in reviews to extract less of the lipids when compared with the chloroform-methanol extraction method. In addition using methanol or hexane-isopropanol may not be safe with the Ultra-turrax
The homogenizing part for Popovic et al seems safe with PMSF but I do not know if these solvents extract the lipids. The article only says it is an inhibitor of protease. Could you take a look?
Chloroform OK if you do this in a fume-hood. Not particularly flammable but can put you to sleep.
David Kessel Thanks for your advise. Wrote to Popovic. they replied to say they used chloroform-methanol 2:1 v/v) in addition to BHT.
This clarifeis all.
Cheers
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