Plant samples contain a complex mixture of proteins with varying properties, making the preparation of high-quality protein extracts a critical step for successful two-dimensional electrophoresis (2-DE) analysis.
The first step in the preparation of plant protein samples is the selection of tissue type and the age of the plant. Plant tissues differ in their protein content and properties, and the age of the plant can affect protein abundance and composition. Younger plant tissues typically have a higher protein content and are therefore preferred for protein extraction.
Select tissue, then, protein extraction is typically performed using a buffer that contains detergents and chaotropes. Detergents help to solubilize membrane-bound proteins, while chaotropes disrupt protein-protein interactions and aid in protein denaturation. The choice of extraction buffer depends on the specific proteins of interest and their physicochemical properties.
To further increase protein solubility, plant protein extracts are often treated with reducing agents such as dithiothreitol (DTT) or beta-mercaptoethanol. These agents break disulfide bonds, which can increase protein solubility and prevent protein aggregation.
Protein extracts may contain impurities that can interfere with downstream analysis, such as salts, lipids, and nucleic acids. Therefore, sample clean-up is often necessary to remove these contaminants and improve protein purity. Common clean-up methods include acetone precipitation, TCA/acetone precipitation, and cleanup with commercial kits.
After the protein extract is purified and solubilized, it can be subjected to 2-DE analysis. During 2-DE, proteins are first separated by isoelectric point using isoelectric focusing (IEF), followed by separation by molecular weight using SDS-PAGE. The separated proteins can then be visualized by staining with a protein dye, such as Coomassie Blue or silver stain.
To identify the proteins on the gel, mass spectrometry (MS) analysis is commonly used. Tandem MS (MS/MS) is often performed to obtain peptide sequences that can be used to search protein databases for protein identification.
1 Extraction From Whole Plant Tissue
a) Plant tissue samples should be ground to a fine powder with a pestle and mortar prechilled in liquid nitrogen.
b) Determine the weight of a 1.5-mL Eppendorf tube before cooling it in liquid nitrogen.
c) Place the powder (from step 1) into the Eppendorf tube and weigh again.
d) Add solution A to the powder for protein extraction. The solution to powder ratio of 1 to 2 (v/w) should be adequate. Vortex immediately to mix the powder with the solution.
e) Add solid protamine sulfate (1 mg/mL).
f) Incubate the mixture on ice for 15 min with occasional vortexing.
g) Centrifuge (13,000g at 2°C) for 15 min. Transfer the supernatant to a new Eppendorf tube and then add solid urea to a final concentration of 9 M. For 5 min, vortex the mixture intermittently at room temperature.
h) The protein-urea extract can be used immediately or divided into 70-μL aliquots, which should be stored at –80°C until required for the first-dimensional IEF gel electrophoresis.
2 Extraction From Intercellular Fluids
2.1. Collection of Intercellular Fluids
a) Immerse entire leaves (100 mg to 1 g, fresh weight) in distilled water in a beaker placed inside a desiccator connected to a vacuum pump. Vacuum filtration of leaf materials is judged finished when the leaf blades have shown a glossy dark green appearance.
b) Gently blot the leaves dry using paper towels before placing the leaves in a 2.5-mL plastic syringe barrel, which should be sitting on top of an Eppendorf tube (with its lid cut off). This whole syringe–Eppendorf tube assembly is then placed inside a 30-mL centrifugation tube.
c) Centrifuge (3000g at 4°C) for 10 min. The intercellular fluid is collected in the Eppendorf tube. It can be used immediately or stored at –80°C until required for the first-dimensional IEF gel electrophoresis.
2.2. Protein Extraction
a) Add 5 vols of solution B to 1 vol of intercellular fluid (see Notes 4 and 5). Incubate the mixture at –20°C overnight.
b) Centrifuge (15,000g at 4°C) for 15 min. Discard the supernatant and dry the pellet under vacuum at room temperature.
c) Resupend the pellet with at least 50 μL of solution C. Vortex vigorously and incubate on ice for 30 min.
d) Centrifuge (14,000g at 4°C) for 15 min. Discard the insoluble material; aliquots of the supernatant should be used immediately and loaded directly onto first-dimensional IEF gels.
You may be interested in:
Reference
- Walker, J. M. (Ed.). (2005). The proteomics protocols handbook. Humana press.