Hi Agata,

Do you this paper :Simões et al. BMC Genomics 2013, 14:181

http://www.biomedcentral.com/1471-2164/14/181.

They work with brain samples and Trizol extraction .They also make sandwich Elisa with their samples .

May be that can help you .

But could you have heavily diluted your resuspended pellet (100-1000 X) in the Elisa buffer to do your test?

Good to you

Vincent

Greetings,

Unfortunately, in my experience, proteins extracted using TRIzol tend to be denatured. It might b realted with the chaotropic activity of the guanidyl group of guanidine thiocyanate in TRIzol. I'd rather isolate both fractions by using ultracentrifugation density gradient, in such a way you can use a buffer that assures you that the proteins would be, at least, in a soluble form.

Good luck

Thank you both for your advice .

Agata

For samples that already in TRIzol the most comprehensive resource I have found is this: (https://www.interchim.fr/ft/D/DU1295.pdf ) . However, I have found that there are 3 key points.

#1 (& most important) DO NOT LET THE PROTEIN PELLET DRY OUT. Aspirate the supernatant and then IMMEDIATELY resuspend in your solubilization buffer (even if there is still some supernatant left). I have found that 2x Laemmli Loading Buffer with beta-mercaptoethanol works superbly for resuspending TRIzol protein pellets for Westerns.

#2 Ensure your sample is appropriately diluted in TRIzol, if you use too little TRIzol or try to precipitate large amounts of protein you will end up with a rubbery pellet that is impossible to resuspend.

#3 Use the DNA Back extraction (pg 18) otherwise I have lost protein using the "classic DNA isolation protocol".

Note 1 : Tip #1 is important in any type of protein precipitation I have ever done, acetone, TRIzol, etc.

Note 2: Resuspension still requires some effort, usually I vortex samples well, let them boil at 95 C for 10-15 min, vortex well again, then if I can still see something you can use a probe sonicator to get the last bit.

Note 3: If you need protein concentrations the Qubit assay works even with loading buffer and beta mercaptoethanol. If you don't have a Qubit, you can make a 2x loading buffer without bromophenol blue or mercaptoethanol for resuspension, measure concentration then add in the bromophenol blue or mercaptoethanol at the end.