the principle would be to have a representative mixture of intact proteins free of contaminants. In principle this would require:

1. extraction of proteins from homogenate to a buffered phenol phase (usually with 2-3 back extractions to increase the yield)

2. sequential precipitation of proteins from the phenol phase using 2-3 different precipitants

Sorry I did not finish before. I will try to attach an example protocol that we use for Arabidopsis roots but if you look it up you will see very little deviation when working with different material. The principle is the same. So attached please find a link to a paper optimizing the whole procedure for plant tissues including precipitation protocols. I hope it will be useful

http://onlinelibrary.wiley.com/doi/10.1002/jssc.200800087/pdf

The protocol is depends on source of the lysate. How ever, addition of TCA/Acetone to lysate and cold storage (-20 C) is an effective precipitation method. For details of protocol, check the following paper:

Comparison of protein precipitation methods for sample preparation prior to proteomic analysis

Journal of Chromatography A, 1023 (2004) 317–320

Refer

http://expasy.org/

I have been dealing with protein precipitation from various samples plant, bacteria, insect and mammals for a while. Although TCA/acetone is the most widely used protocol, I recommend ammonium-acetate/methonaol- acetone precipitation. The main pitfall of TCA/acetone is that TCA is highly acidic and even after several washes with acetone, it is difficult to remove the complete acidic effect of TCA. This hinder resolubilization of TCA/acetone precipitated proteins. Based on this problem, scientist came up with an idea of increasing pH with NaOH before resolubilization. On the other hand, ammonium-acetate/methonaol- acetone precipitation method proteins are precipitated at higher pH (8.0) which leads better resolubilization of the precipitated proteins. Hope this helps.

Nazrul, could you please refer to a detailed protocol for ammonium-acetate/methanol-acetone precipitation ?

Hi, I agree with Reza, TCA/acetone protein precipitation gives a very clean protein sample, then the solubilization of precipitate with urea-based buffer (proper for elechtrophoresis, such as IEF) neutralizes the acidity of TCA. It works well, that's my experience.

I do agree with Maria that there is no problem in dissolving TCA/acetone clean protein with urea based buffer compatible with IEF. But if you are not doing IEF, for instant you want to run SDSPAGE, then I believe ammonium-acetate/methonaol- acetone precipitation would be a good option.

Sorry my question is coming 5 years later. Nazrul, can you share the protocol for ammonium-acetate/methonaol- acetone precipitation? Thank you!