Maize Protein Extraction for Different Downstream Applications

Sample preparation is a key step for making plant proteomics analyses reproducible. The large amount of organic compounds, complex polysaccharides, proteases and other cellular components in vascular plants interfere with the analysis of proteins from these organisms (Carpentier et al., 2005; Wang et al., 2008). This makes the development of suitable methods for this a challenge and makes key steps in plant protein extraction including tissue disruption, secondary metabolites removal and protein solubilization even more critical. Nonetheless, the downstream application is the most important thing that a researcher needs to consider when choosing a protein extraction method. Maize has been utilized as a model plant for developmental genetics in the grasses, and researchers working with maize have been applying molecular techniques to be able to analyze proteins in planta at similar to how proteins are analyzed in eudicot model plants. Here, I describe a simple maize protein extraction method using different extraction buffers that can be used for simple downstream applications like immunoblot as well as more complex applications like immunoprecipitation (IP). One advantage of this method is that it is very fast, so there is an increased chance of being able to analyze unstable proteins and proteins found in low abundance by immunoblot and/or IP. Another advantage of this protocol is that it does not require subcellular fractionation, which may degrade unstable and/or unabundant proteins. Depending on the downstream application to be used, this protocol is flexible and it is compatible with buffers that contain detergents and chaotropic agents which result in varying degrees of protein denaturation. In the recipes section, buffer preparation instructions go from milder conditions to more denaturing conditions. Buffers 1 and 2 are suitable for immunoprecipitation of protein complexes since they are milder buffers and many protein-protein interactions will be preserved in these buffers. Buffers 3 and 4 are suitable for immunoblot detection (Western blot) since they use ionic detergents and reducing reagents that reduce protein disulfide bonds. If the protein extract will be analyzed for the phosphorylation status of a target protein, then phosphatase inhibitor mix can be added to the recipes.

Maize

Protein

Extraction buffer

Immunoblot