Plant whole protein extraction by trichloroacetic acid-acetone method
Plant whole protein extraction by trichloroacetic acid-acetone method
In this chapter we describe an efficient method suitable for whole protein extraction from various plant tissues. The method utilizes trichloroacetic acid and acetone containing mercaptoethanol to denature proteins while precipitating them. Proteolysis methods are also described prior to classical IEF electrophoresis or electrophoresis with IPG adhesive strips, respectively. The source of this experiment is the "Guide to Plant Proteomics Experiments" [French] H. Tillemment, M. Zivi, C. Damerweiler, V. Mitschine, eds.
Operation method
Plant whole protein extraction by trichloroacetic acid-acetone method
Materials and Instruments
Plant Move 1. Protein precipitation and denaturation For more product details, please visit Aladdin Scientific website.
TCA β-mercaptoethanol acetone R2D2 Protein Lysis Buffer UKS Protein Lysis Buffer IPG Glue Strip Hydration Solution
Mortar and pestle
( 1 ) Plant material is ground to a fine powder in liquid nitrogen using a mortar and pestle (see Note 4).
( 2 ) About 200 μl of the powder is transferred to a 2 ml centrifuge tube and 1.8 ml of pre-cooled trichloroacetic acid-2ME-acetone solution (see Note 5) is added and the mixture is placed at -20°C for 1 h. TCA and acetone denature proteins and precipitate them, and inactivate polyphenol oxidases and other oxidative enzymes, thus preventing the formation of insoluble complexes by combining proteins with each other as a result of phenol oxidation to wakes. This prevents proteins from combining with each other to form insoluble complexes due to the oxidation of phenols to wakes. This method inactivates proteases in the same way as phenol extraction [7,8] [3]. Acetone also solubilizes various pigments, lipids and terpenoids in plant cells. 2ME prevents the formation of disulfide bonds during protein precipitation.
( 3 ) Centrifuge at 10,000 g for 10 min.
2. 2ME-acetone solution for washing protein samples
( 1 ) Discard the supernatant obtained by centrifugation and suspend the precipitate in 1.8 ml of pre-cooled washing solution (see Note 5). -The purpose of the wash is to remove residual TCA and to avoid the difficulty of protein reconstitution and solubilization due to acidification.
( 2 ) Centrifuge at 10,000 g for 15 min.
( 3 ) Discard the supernatant and repeat the washing operation twice (see Note 6).
( 4 ) Dry the precipitate under vacuum for 1 h (or in a SpeedVac for 20-30 min) to completely remove residual acetone.
( 5 ) Weigh the precipitate (see Note 7).
3. Protein solubilization
( 1 ) The amount of UKS or R2D2 required to solubilize a protein sample depends on the plant tissue. For example, we solubilize protein samples from corn and grape leaves at 60 μl/mg dry powder, and protein samples from corn kernels at 50 μl/mg dry powder.
( 2 ) The dry powder sample was re-dissolved by shaking for 1 min. At this point, the sample contained cellular debris.
( 3 ) Centrifuge at 10000 g for 15 min at room temperature and collect the supernatant in another centrifuge tube.
( 4 ) Centrifuge again for 15 min. The supernatant is the resulting protein sample and can be stored at -50 or -80°C for several months.
Generally, solutions used to suspend and solubilize protein samples contain dissociating, denaturing, and reducing agents [9] . Dissociators help to defold proteins by breaking the non-covalent bonds within the protein. The most commonly used dissociators are urea and thiourea. They are often used in combination to increase the solubilization efficiency of proteins. A denaturant must be used in conjunction with a dissociative agent to increase the solubilization of proteins CHAPS is a partially ionic denaturant that has the same solubilizing ability as the nonionic denaturant Triton X-100, but it is more effective at preventing protein interactions SB3-10 has a stronger solubilizing ability than CHAPS but it is difficult to solubilize in solutions with a high concentration of urea. SB3-10 has more solubilizing power than CHAPS, but it is difficult to dissolve in solutions with higher concentrations of urea. Sulfhydryl reducing agents (most commonly DTT) and phosphine hydride prevent the formation of disulfide bonds. Phosphine is more selective and efficient than DTT, and it retains its reducing power under acidic conditions and at pH greater than 7.5. It is often stored as TCEP-HCl (triscarboxyethyl phosphine), which is more stable, less volatile, and more soluble in water than the preserved form of TBP.
The R2D2 Protein Lysis Solution is suitable for isoelectric focusing with IPG tape and is prepared using a combination of urea and thiourea (see Note 2), CHAPS and SB 3-10, DTT and phosphine.
The UKS solution was a protein solubilization solution often used in the early days before it was discovered that thiourea and concurrent ionic denaturants could greatly increase protein solubilization. This solution contains a high concentration of urea (nearly saturated, see Note 2) and uses Triton X-100 rather than CHAPS and SB 3-10. UKS is characterized by the presence of the ionic denaturant SDS, which greatly enhances protein solubilization and inhibits protease activity [10]. Although the ionic denaturant SDS is not compatible with the isoelectric focusing buffer system, the buffer system can tolerate the concentration of SDS because Triton X-100 can form colloidal ions with SDS and move to the cathode during electrophoresis, and the buffer system can be alkalized by K2CO3, which organizes the interaction between proteins and proteins as well as proteins and nucleic acids, and inhibits protease activity. protein-protein and protein-nucleic acid interactions and inhibits protease activity.
R2D2 can be used to both solubilize protein samples and hydrate IPG film, whereas when using protein samples solubilized by UKS, a separate IPG film hydration solution must be used. The protein solubilizing capacity of the two solubilizing solutions is roughly equivalent, so it is recommended that only one of them is used. UKS is recommended if the sample being prepared is likely to have a high amount of protease, and the addition of amphoteric electrolytes to both solutions improves the resolving power of isoelectric focusing electrophoresis.
4. Preparation of protein samples for IEF electrophoresis
( 1 ) Protein samples need to be centrifuged again to remove the precipitate before electrophoresis.
( 2 ) If silver nitrate staining method is used, the sample volume of each strip (24cm long, homemade glue or IPG glue) is about 50 μg of protein (Fig. 1-1A, B); if CBB staining method is used, it is 150~500 μg (Fig. 1-1C, D).
( 3 ) Protein samples dissolved in R2D2 can be used to hydrate IPG film directly after the volume of R2D2 solution has been made up to 450 μl [ 6] .
Samples dissolved in UKS can be used directly for conventional isoelectric focusing electrophoresis [3]. If IPG adhesive strips are used for isoelectric focusing, they must be hydrated with a separately prepared hydration solution (Figure 1-1A and B, see Note 8) to a volume of 450 μl. The separately prepared hydration solution contains thiourea, CHAPS, and phosphorus trihydroxide, and also reduces the concentration of SDS and improves resolution.