17 December 2020 1 7K Report

So - we are occasionally using methanol/ammonium acetate precipitation of proteins - from phenol or aqueous solution. Essentially as described in -

Faurobert, M., Pelpoir, E. & Chaïb, J. Phenol Extraction of Proteins for Proteomic Studies of Recalcitrant Plant Tissues. in Plant Proteomics: Methods and Protocols (eds. Thiellement, H., Zivy, M, Damerval, C & Méchin, V.) vol. 355 9–14 (2007).

This chapter references - Hurkman, W. J. & Tanaka, C. K. Solubilization of Plant Membrane Proteins for Analysis by Two-Dimensional Gel Electrophoresis. Plant Physiol 81, 802–806 (1986).

And you can find e.g. Wu, X., Xiong, E., Wang, W., Scali, M. & Cresti, M. Universal sample preparation method integrating trichloroacetic acid/acetone precipitation with phenol extraction for crop proteomic analysis. Nat Protoc 9, 362–374 (2014).

And other references to this approach can be found.

Now, what I am missing (and maybe I am going nuts and overlooked it in these papers, somehow) is the significance of ammonium acetate here - it is a natural pH 7 buffer (at least in water) and pH control is a good thing in most cases - and I believe I read somewhere (but can't find where now) that alcohol (organic?) precipitations of proteins are most efficient/reproducible at near neutral pH - is this an isoelectric point consideration for an 'average' protein (in which case I thought slightly acidic would be better based on 'average' pI [disclaimer - in aqueous solution]).

Then I came across this:

Nickerson, J. L. & Doucette, A. A. Rapid and Quantitative Protein Precipitation for Proteome Analysis by Mass Spectrometry. J Proteome Res 19, 2035–2042 (2020).

And it made me consider if the contribution of the ammonium acetate is the ionic strength it imparts to the solution (or, ionic strength + neutral pH buffering together).

I am still on the hunt for the reference I believe I read long ago that addressed this matter -- but in the mean time I figured I would solicit feedback from the community to perhaps expedite me arrival back at the correct answer.

More John LaCava's questions See All
Similar questions and discussions