Experiment 1: PROTEIN ISOLATION USING TRIZOL

THEORETICAL FRAMEWORK

Before a protein can be characterized, it must first be extracted from the original

biological source; typically, non–recombinant proteins must first be liberated from their original cellular or tissue location. Protein extraction and purification usually involves disruption of cells and tissues containing the target proteins, and removal of non-protein substances. Extracted proteins can then be further purified via solvent extraction or precipitation.

Plant protein extraction is relatively more difficult to perform compared to extraction from

other biological samples (e.g., bacterial cells, mammalian cells and tissues). Unlike other types

of cells and tissues, plants contain a lot of polysaccharides (i.e. cellulose), lipids, and polyphenols that require additional steps to remove. Structural rigidity of some parts of the plants increases the difficulty in homogenizing and permeabilizing the tissues. Also, the amount of proteins available in plant tissues is low relative to other biomolecular components. Therefore, plant protein extraction should be done carefully and systematically.

REAGENTS

TRIZOL® Reagent Chloroform Absolute Isopropanol 0.3 M guanidine hydrochloride in 95% EtOH Absolute Ethanol 1% SDS

PROCEDURE

1. PREPARATION OF PLANT SAMPLES

a. Prepare plant samples (mongo beans) in different stages: seeds, sprouts (1-2 days), and adults (3-4 days). For the sprouts and the adults, plant the seeds on cotton placed in a sturdy container, then water the plants at least once a day with 5 mL of the prepared watering solution.

b. Place the fresh samples in a stainless steel mortar and pestle, and grind. Make sure that samples are placed on ice while grinding.

c. Weigh approximately 0.1 g of the sample in a pre-sterilized microcentrifuge tube (MCT). Samples may be stored at -20 °C for a month or -80 °C for longer storage.

2. HOMOGENIZATION

a. Homogenize tissue samples in 1 mL of TRIZOL® Reagent per 50-100 mg of tissue. The sample volume should not exceed 10% of the volume of TRIZOL® Reagent used for homogenization.

b. Remove insoluble material from the homogenate by centrifugation at 12,000 × g for 10 minutes at 2 to 8°C. Transfer the cleared homogenate solution to a

fresh tube.

3. PHASE SEPARATION

a. Incubate the homogenized samples for 5 minutes at 15 to 30°C.

b. Add 0.2 mL of chloroform per 1 mL of TRIZOL® Reagent. Cap sample tubes securely.

c. Shake tubes vigorously by hand for 15 seconds and incubate them at 15 to 30°C for 2 to 3 minutes.

d. Centrifuge the samples at no more than 12,000 × g for 15 minutes at 2 to

8°C.

e. Following centrifugation, the mixture separates into a lower red, phenol-chloroform phase, an interphase, and a colorless upper aqueous phase.

f. Discard the aqueous phase.

4. DNA PRECIPITATION

a. To the lower red, phenol-chloroform phase and interphase, add 0.3 mL of 100% ethanol per 1 mL of TRIZOL® Reagent used for the initial homogenization, and mix samples by inversion.

b. Store the samples at 15 to 30°C for 2-3 minutes and sediment DNA by centrifugation at no more than 2,000 × g for 5 minutes at 2 to 8°C.

c. Discard the pellet and save the phenol-ethanol supernatant

5. PROTEIN PRECIPITATION

a. Add 1.5 mL 100% isopropanol per 1 mL TRIZOL® Reagent used for the initial homogenization.

b. Store samples for 10 minutes at 15 to 30°C, and sediment the protein precipitate at 12,000 × g for 10 minutes at 2 to 8°C.

6. PROTEIN WASH

a. Remove the supernatant and wash the protein pellet 3 times in a solution containing 0.3 M guanidine hydrochloride in 95% ethanol. Add 2 mL of wash solution per 1 ml of TRIZOL® Reagent used for the initial homogenization.

b. During each wash cycle, store the protein pellet in the wash solution for 20 minutes at 15 to 30°C and centrifuge at 7,500 × g for 5 minutes at 2 to 8°C.

c. After the final wash, vortex the protein pellet in 2 mL of ethanol.

d. Store the protein pellet in ethanol for 20 minutes at 15 to 30°C and centrifuge at 7,500 × g for 5 minutes at 2 to 8°C.

7. REDISSOLVING THE PROTEIN PELLET

a. Vacuum dry the protein pellet for 5-10 minutes. Dissolve it in 1% SDS by pipetting.

b. Complete dissolution of the protein pellet may require incubating the sample at 50°C.

c. Sediment any insoluble material by centrifugation at 10,000 × g for 10 minutes at 2 to 8°C, and transfer the supernatant to a fresh tube. The sample may be stored at -5 to -20°C for future use.