Proteomic study of recalcitrant plant tissues (protein extraction by phenol method) Experiments
Another feasible method for protein extraction, besides the classical trichloroacetic acid (TCA ) /acetone precipitation method, is phenol extraction, which can efficiently obtain proteins from plant tissues rich in polysaccharides, lipids and phenolic compounds. This chapter presents a modified experimental method that can be used for bidirectional electrophoresis (2-DE) and further proteomics studies. The source of this experiment is the "Guide to Plant Proteomics Experiments" [France] H. Tillemment, M. Zivi, C. Damerweil, V. Mitchen, eds.
Operation method
Proteomics of recalcitrant plant tissues (protein extraction by phenol method)
Materials and Instruments
Plant tissue Move 3.1 Protein extraction For more product details, please visit Aladdin Scientific website.
Phenol Extraction Buffer Precipitation Solution Isoelectric Focusing Buffer
Cryogenic Pulverizer
( 1 ) Fresh plant tissues are frozen in liquid nitrogen, and the material is then ground to a fine powder in a pre-cooled steel cup in an automated cryogenic pulverizer (see Note 4).
( 2 ) Add 3 ml of extraction buffer to a 15 ml falcon tube with 1 g of finely ground plant tissue, vortex and shake in an ice bath for 10 min (see Note 5).
( 3 ) Add an equal volume of Tris saturated phenol and shake for 10 min at room temperature (see Note 6).
( 4 ) Centrifuge the sample at 5500 g and 4°C for 10 min, separating from top to bottom into the organic phase, the aqueous phase, and the precipitated insoluble material (see Note 7), and carefully transfer the top layer of phenol to a new centrifuge tube, taking care to avoid touching the middle layer.
( 5 ) Add 3 ml of extraction buffer to the protein-containing phenol solution for back- extraction. Shake for 3 min and vortex. The samples were then centrifuged at 5500 g and 4°C for 10 min.
( 6 ) Transfer the upper phenol phase to a new centrifuge tube and add 4 times the volume of precipitate. Shake the tube upside down and allow to settle at -20°C for at least 4 h, or overnight.
( 7 ) Centrifuge to precipitate the protein (10 min, 5500g, 4°C).
( 8 ) After centrifugation, the precipitate is rinsed three times with pre-cooled precipitation solution and finally with pre-cooled acetone. After each rinse, the samples were centrifuged (5 min, 5500 g, 4°C).
( 9 ) Vacuum dry the precipitate (see Note 8).
The proteins are first extracted into Tris buffer containing several protective agents: EDTA inhibits metalloproteases and polyphenol oxidases by chelating metal ions; PMSF irreversibly inhibits serine proteases; and β-mercaptoethanol is an inhibitor of β-mercaptoethanol. mercaptoethanol is a reducing agent that prevents oxidation of proteins. In addition, in order to inhibit the protease activity, the experimental temperature must be kept below 4°C and the first step of the extraction should be carried out in an ice bath. The extraction time is minimized. KCl is used because of its salt solubility effect, which can increase protein solubility and facilitate protein extraction.
In contrast to the traditional phenol extraction method, Wang et al. proposed another feasible method called phenol/SDS extraction due to the addition of sodium dodecyl sulfate (SDS). This method significantly increased the amount of protein extracted from olive leaf tissue, with clearer bi-directional electrophoresis and more concentrated protein spots than phenol alone. However, the addition of SDS did not improve protein extraction from banana, apple and potato leaves.
Tris buffer containing sucrose is denser than Tris saturated phenol. Therefore, when the phases are separated, the phenol phase can be separated to the uppermost part of the centrifuge tube more quickly, which facilitates the transfer of the phenol phase (see Note 7). In the upper part the phenol phase contains cytoplasmic and membrane proteins.
A Tris saturated phenol solution of pH 8.0 ensures that nucleic acids are separated into the buffer phase without leaving the nucleic acids in the phenol phase.
Proteins are usually separated by the addition of salts or water-soluble organic solvents. Here, both methods were applied.4 Times the volume of methanol was effective in separating most of the proteins. However, methanol is less effective in extracting proteins from acidic solutions. The use of organic bases or buffers (ammonium acetate) can solve this problem.
3.2 Solubilization and quantification of proteins
( 1 ) The protein precipitate is resuspended in IEF buffer. If the starting material is 1 g of fresh tomato fruit tissue, 200 μl of IEF buffer is required.
( 2 ) The sample should be shaken at room temperature for at least 1 h (sometimes longer). Do not heat the sample as this will result in carbamylation of the proteins.
( 3 ) For quantification, some ovalbumin standards (8 dilutions from 0 μg/μl to 60 μg/μl) are diluted with IEF buffer. Then, 10 μl of 0.1 mol/L HCl was added to each dilution, and finally, the sample tubes, whether used for the standard curve or for the determination of the sample concentration, were volume-filled to 100 μl with distilled water.
( 4 ) Then 3.5 ml of diluted stain was added and the absorbance value of the sample solution was read at 595 nm.
For the determination of protein concentration in plant tissues, the Caulophylline blue method [9] is more suitable than the Lowry method [10] and the Biuret method because the latter two are based on the quantification of phenolic compounds [1]. However, it is not easy to quantify directly in the sample buffer due to the interference of IEF buffer components. Therefore, we adopted the modified Ramagli and Rodriguez method, which is based on the acidification of the sample buffer and can be used for the direct quantification of proteins dissolved in the sample buffer even if the buffer contains urea, carrier ampholytes, nonionic detergents, and thiols.