I think that it is better if you use acetone precipitation or ammonium sulphate fractionation. About the non-precipitation of proteins, I think that it is due to the low concentration of acetone in the sample. Indeed, usually a final concentration of 80% acetone is necessary for the protein precipitation.

This is the procol for the precipitation with acetone:

- cool the acetone at -20°C.

-place the protein solution in a tube compatible with acetone.

-add four times the sample volume of cold acetone (e.g. 4 ml of acetone +1 ml sample)

-mix well and incubate for 60 minutes at -20°C

-centrifuge 10 minutes at 13000xg

-discard the supernatant and then let the pellet dry a bit in the air (usually 30 minutes and NOT longer, otherwise the pellet might not dissolve and the proteins might loose the activity).

-add the buffer of choice and mix well until the pellet is dissolved.

I hope this might help you!

Ammonium Sulphate is the best option to precipitate protein because I also precipitated my plant protein using 60% ammonium sulphate. You may use 80% ammonium sulphate according to your protein requirement.

Dear Rahul

TCA/acetone is the best method for protein precipitation because approximately all proteins are precipitated and all interfering materials are removed. This method is easy and can be performed in all labs.

If you need this procedure, please dont hesitate to contact me.

Thanks Masood for helping me. 

Actually i want to precipitate plant protein. So is the TCA/acetone method is suitable? 

Thanks Sobia.

If u provide me your protocol, this can be helpful to me.

Yes, the TCA/acetone method is suitable for plant protein.

You can find it usage in several studies:

1. Electrophoresis, 27(13), 2782-2786.

2. Electrophoresis 8.2 (1987): 93-99.

3. Plant Proteomics 2007. 1-8.

and 28 newest article publish 2015.

Dear Masood,Thanks for your instruction.

i will follow your given protocols.   

Rahul,

What are you using the Lowry Assay for? Is it to do equal loading on your gel? You are using 100% acetone to ppt the protein, did you make an appropriate dilution to bring the % acetone into compatibility with the Lowry assay? The Lowry assay is only compatible to 10% acetone. If you are equal loading based on the Lowry assay quantitation at 100% acetone it is possible you may have over diluted the sample prior to gel loading and will not see much of anything on the gel. This can be further compounded by the type of staining technique you are using, coomassie blue is less sensitive then silver stain.

Hello, According to my experience in microbial protein, in relation to the protein precipitation, I have obtained good results using 3 volumes of acetone, if I added 20% TCA is better. The major problem is the protein hydration after precipitation. I advise you to do it in Tris buffer. However, if you want to make two-dimensional gels this buffer does not work, then I advise you the ultrafiltration method, there are some comercial filters very useful.

I think  Ammonium Sulphate is a good  protocol. I did use it with very good results. My proteins were 100-150 kDaltons.

Greetings. 

As far as I know, once you precipitate the protein with cold acetone, you have to centrifuge it in order to collect the pellet and then let the pellet dry a bit to get a rid of acetone. Then you can try soluilize the pellet. I said try because sometimes it can be tricky to solubilize it. If you are using TCA (final concentration in the sample at least 10%, let the sample stay in ice for 30 minutes), you can solubilize the pellet easily by adding a basic buffer (like tris pH 8-9) or just by adding concentrated NaOH (you need to change the pH in order to displace the TCA from the complex TCA-protein). These procedures works well you don't need a really functional protein (I mean, if you are not studing enzyme activities). If you need a functional protein, you can use the acetone precipitation (you have to expect a loose in activity) or ammonium sulphate fractionation.

Dear Alessandro,

I want to study the functional activity of extracted protein. So according to you what procedure is suitable? 

Dear Cindy, 

I tried to precipitate  the protein using 10% acetone. After adding equal volume of acetone in extract, left the tube overnight in 4oc temp. Next day i don't see any precipitation of protein. 

I think that it is better if you use acetone precipitation or ammonium sulphate fractionation. About the non-precipitation of proteins, I think that it is due to the low concentration of acetone in the sample. Indeed, usually a final concentration of 80% acetone is necessary for the protein precipitation.

This is the procol for the precipitation with acetone:

- cool the acetone at -20°C.

-place the protein solution in a tube compatible with acetone.

-add four times the sample volume of cold acetone (e.g. 4 ml of acetone +1 ml sample)

-mix well and incubate for 60 minutes at -20°C

-centrifuge 10 minutes at 13000xg

-discard the supernatant and then let the pellet dry a bit in the air (usually 30 minutes and NOT longer, otherwise the pellet might not dissolve and the proteins might loose the activity).

-add the buffer of choice and mix well until the pellet is dissolved.

I hope this might help you!

Well, if you want to check the functional activity of your proteins then avoid TCA precipitation. In fact, TCA partially denatures the proteins and the denatured proteins may lose their functional activity.