Dtt

50 mM 71.5 mg

Autoclaved MQ

final vol. 100 mL

FIGURE 11.4. Composition of the TCAEEB solution, the wash buffer, and the LB-TT (resolubi-lization of pellet and rehydration/focusing solution) used for rice leaf protein extraction.

FIGURE 11.4. Composition of the TCAEEB solution, the wash buffer, and the LB-TT (resolubi-lization of pellet and rehydration/focusing solution) used for rice leaf protein extraction.

is given in Figure 11.4. Recent results also demonstrate that the TCAAEB/LB-TT protocol can significantly improve the solubilization of proteins from not only rice tissues, but also from Arabidopsis, maize, cucumber, and wheat seed embryos [13]. Researchers also have a wide choice of buffers to use and adapt for a particular sample, as evident from many research publications. In this regard, we also recommend using the LB-TT (direct extraction) or the phenol buffer. The former gives excellent extraction efficiency while avoiding loss of proteins, while the latter is useful for removing contaminants (during IEF) such as nucleic acid, phenols, polysaccharides, and so on. Ultimately, however, a researcher can use the extraction protocol of their choice by modifying and adapting available protocols to extract the best quality and composition protein, free of contaminants.

The second critical step for developing a proteome reference map (via 2D gel analysis) is the focusing and separation of extracted protein in both the first and second dimensions. Although 2-DGE has inherent drawbacks, such as underrepresentation of highly acidic, basic, hydrophobic, and extremely high- and/or low-molecular-weight (HMW/LMW) proteins, it is the method of choice for most laboratories (its merits/demerits are further debated in Chapter 2). In rice, the hand-cast IEF system has long been the method of choice for many research groups mainly due to cost-effectiveness [3, 4]. We feel that a shift to pre-cast IPG technology and large-format gels is a necessity. Most laboratories and research groups focused on rice proteome research, especially those developing high-quality 2D reference gel maps, are already using this technology [4]. Furthermore, we have standardized the IEF protocol for IPG strip formats (11-24 cm in length; GE Healthcare) using rice leaf proteins as an example (this is presented in Figure 11.5). This protocol can be further modified according to each specific sample and condition if required. With the use of a 24-cm IPG strip (pH 4-7), ~910 protein spots stained with silver nitrate (for a standardized staining protocol, see Table 11.1) were detected in rice leaves on pre-cast 12.5% DALT large-format PAG (Figure 11.6). The pre-cast IPG strips are therefore recommended not only for the better resolution of protein spots but also for greater reproducibility in generating high-quality 2D gel reference maps [4, 14].

Once a high-quality 2D gel has been obtained, the last two steps in the completion of a proteomics study are image analysis (see Chapters 2 and 8) and protein identification. For identifying the stained proteins spots, one can use either N-terminal amino

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