Tim Fallon, PhDJun 26, 2023

Shout out to #protein #biochemistry world. I'm trying to do a protein isolation from freeze dried tissue. In my last attempt, fats & pigments (it's not a mammal) gummed everything up.

Does anyone have any first principles downsides to a pre-extracting with ethyl acetate (i.e. "defatting") to remove the fats & pigments? Since my protein is already dry / precipitated in some form due to the freeze drying, I can't imagine extracting it with a chemically inert organic solvent could really hurt?

Show threadTim Fallon, PhDJun 26, 2023
Yes this is a lot like the "acetone powder" extraction that #biochemists of old used to do... But, I'm a little skeptical of acetone, for perhaps unjustified reasons (potential covalent modifications? nucleophilic attack against the carbonyl? nucleophilic attack by Claisen condensation?). Rationalizing that ethyl acetate is more chemically inert, and still (slightly) miscible with H2O like acetone is.
Show threadTim Fallon, PhD

This paper suggests acetone (at room temp) does modify peptides: both at α-amino or ε-amino groups of a peptide, as well as a poorly understood but rapid modification at glycine residues in a specific sequence context.

https://doi.org/10.1021/pr900806x

Jun 27, 2023 at 1:39amWeb