See attached paper.

• There are some protein extraction methods (trichloroacetic acid [TCA]/acetone, Mg/NP-40/TCA, and tris-base/acetone) . The TCA/acetone method exhibited clear protein profiles and detected more protein spots with the highest intensity in the region of high Mr (above 45 kDa) than the other methods

Hi dear Ayu Zahrotul Fuadati ,

I recommend this book chapter to you. You will also find the whole book useful.

Article Protein Extraction from Plant Tissues

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Egbuna

See attached paper.

For extraction of protein from plant sources, use fresh leaves. Going with dried leaves leads to denaturation of a protein. The TCA and ammonium sulfate precipitaion gives clear profiles. In protein extraction dialysis is an important step because sometimes precipitation affects the solubility of the extracted protein..

i copied some where but hope it will help...

Provide a well buffered environment to avoid sudden changes in the pH. Most commonly used biological buffers (phosphate, Tris, MOPS and HEPES) have pKa near 7 so they can be used at physiological pH.

Carry out the extraction at the right temperature. While proteins from mammalian and bacterial cells can be extracted at 37oC, plant proteins should be extracted at a much lower temperature (4oC) to reduce the activity of the proteases present in the solution.

Use the right amount of the right additives (salts, protease/peptidase inhibitors, osmolytes and reducing agents) to aid in the extraction process.

Protein Precipitation, Why?

Basically, protein precipitation is carried out for two reasons - to separate the proteins from any unwanted contaminants and to concentrate the protein yield. In most cases, protein extraction yields a large volume of dilute protein since a large amount of starting material is usually required to harvest an adequate amount of a protein.

To precipitate proteins, hydrophobic aggregation is encouraged through either one of these methods:

Subtle disruption of the folded structure of the protein to expose more of the hydrophobic interior to the solution

Dehydration of the shells of water molecules forming over hydrophobic patches on the surface of properly folded proteins

Once the proteins aggregate into larger structures, the amount of water per protein is greatly reduced and the density differences between the proteins and the solute is enhanced significantly. If there are enough aggregates large enough to disrupt the path of light through the solution, they can be pelleted with the application of centrifugal force. The extra solvent and other unwanted contaminants are then removed to make them ideal for downstream applications.

Protein Precipitation Protocols

Acetone/TCA precipitation

Protein precipitation can be achieved by using trichloroacetic acid (TCA) to alter the pH of the medium. By adding TCA to the solution, the electrostatic forces are significantly reduced, thereby exposing more of the hydrophobic structure of the proteins. Organic solvents such as acetone are used in conjunction with TCA to reduce the dielectric constant of an organic solvent and decrease the solubility of the protein. Since this method uses an acid, it requires the addition of a base and/or washing off the remaining protein with acetone after the precipitation process has been completed.

Mix 10 volumes of cold 10% TCA in acetone (stored -20oC) with your samples. Vortex and incubate at -20oC for at least three hours (overnight incubation is preferable).

Centrifuge samples at 15000 x g for 10 minutes. Discard supernatant carefully to avoid disturbing the pellet.

Add the same volume of cold acetone. Vortex, and let stand at -20oC for at least 10 minutes to remove the acid.

Centrifuge at 15000 x g for five minutes, remove supernatant and allow pellets to air dry. Do not allow complete desiccation of the protein pellet since this will make re-solubilization more difficult.

Note: While this is a standard method for protein precipitation, this protocol is capable of denaturing proteins. Thus, it is usually used for applications that do not need an active protein (e.g. in precipitating contaminant proteins). It is also ideal when working with dilute samples.

TCA/DOC precipitation

Sodium deoxychelate (DOC) is an anionic bile acid detergent that works effectively in disrupting and dissociating protein interactions, as well as in facilitating protein precipitation.

Mix sample with 1/100 of its volume of 2% DOC. Incubate on ice for 20 to 30 minutes.

Add enough 100% TCA to bring sample to a final TCA concentration of 15%. Mix by vortexing for 30 seconds to prevent the formation of any large conglomerates that may trap the contaminants in the solution.

Allow the sample to precipitate for at least one hour (overnight precipitation would be advisable).

Spin out the precipitate at 15,000 x g for 10 minutes. Aspirate the TCA and soluble contaminants without disturbing the pellet.

Wash pellet with ice cold ethanol or acetone to remove excess TCA. Break up the pellet to make sure it is washed thoroughly.

Vortex and let sit at room temperature for 5 minutes.

Spin down the pellet at 15,000 x g for another 10 minutes and aspirate the supernatant off of the pellet.

Repeat the wash and spin steps again.

Allow the pellet to dry under a slow stream of nitrogen but do not dry it completely since it will be difficult to bring back into solution.

Note: This protocol can be useful for 1D and 2D gel analysis but is not compatible with mass spectrometry.

Chloroform/Methanol precipitation

This method works extremely well for precipitating proteins from various sources, and produces a dry protein material that is both salt- and detergent-free. Here’s how to proceed when using this protocol:

Bring up predetermined amount of protein extract to 100 μl with water.

Add 4 volumes (400 μl) methanol and vortex well.

Add 1 volume (400 μl) chloroform and vortex vigorously.

Centrifuge for two minutes at 15000 x g. The proteins should appear as a thin wafer or a circular flake at the liquid interface. However, there are times when precipitated proteins are not visible.

Remove the aqueous top layer (water-methanol mix) without disturbing the interface.

Add 4 volumes of methanol to wash the precipitate.

Vortex vigorously and centrifuge for two minutes at 15000 x g.

Remove as much supernatant as possible without disturbing the delicate precipitate which adhered to the walls or settled to the bottom of the tube.

Dry under vacuum or nitrogen