Experiment-3: Gel-based Proteomics to Analyze Plant Proteome
To compare the protein expression profiles of plant leaves grown under drought (stress) and normal conditions using 2D gel electrophoresis.
Plants have appreciable amount of complexity with respect to protein networks. Studying the proteome of plants could help in providing a comparative analysis with closely related proteomes of other organisms. Some plants such as Arabidopsis thaliana, which is commonly used as a model organism, are often studied at the proteome level to gain deeper insights into the various biological processes. Some plants have therapeutic values and are often studied to get more information of the protein nature, when exposed to several stress responses. In this section, we have described the procedure for analysis of plant leaf proteome using 2DE. Experimental plan involves the collection of plant leaf, protein extraction followed by separation of proteins on 1st and 2nd dimension. For detail theory of 2DE users are advised to see the Experiment 1.
Preparation of sample:
- Around 300 mg of fresh leaf is collected.
- First of all the leaves must be homogenized properly using a clean mortar and pestle.
- Liquid nitrogen is added to the homogenized leaves to freeze and solidify rapidly. This solidified leaf parts are then mashed thoroughly until a powder is obtained.
- To the powder, 500 µL of lysis buffer (acetone, 10% TCA, 0.07% DTT) is added and the contents are mashed vigorously to prepare a homogenous mixture. More lysis buffer can be added, if required, to make the process efficient. Once it is finely homogenized, the volume is made up to 1.5 mL and this mixture is kept at -20°C for 1 h.
- It is then centrifuged at 14,000 g for 30 min at 4°C. This enables the formation of a pellet of proteins and the supernatant can be discarded.
- Chilled acetone with 0.07% DTT is then added to the pellet and vortexed briefly.
- The mixture is again centrifuged at 14,000 g for 30 min at 4°C, after which the supernatant is discarded. This step is repeated 3 more times.
Figure 1: Protein extraction from the plant leaves. (a) Fresh leaves, clean the leaves properly (b) treat the leaves with liquid nitrogen and start grounding (c) fine homogenous paste prepared using buffer (d) debris precipitated using centrifugationn
- After washing step, the pellet is left to dry at room temperature for about 40 min. Ensure that the pellet is totally dry.
- The dry pellet is then dissolved in 400 µL of rehydration buffer (composition same as mentioned in section 1: C) and vortexed briefly. This is then stored overnight at 4°C for protein extraction.
- Next day, the mixture is centrifuged at 14,000 g for 15 min at 4°C.
- The supernatant containing the proteins are separated carefully and stored in a fresh microcentrifuge tube at -20°C until further use.
Figure 2. Image of a typical 2D gel showing plant proteome separated on a 4-7 pH range IPG strip.