Abstract
Since its introduction in 1979 (Towbin et al., 1979), protein blotting (Western blot) has become a routine tool in molecular biology research. It is used to detect low amounts of proteins in complex samples or to monitor protein expression and purification. Western blotting includes protein complex separation by gel electrophoresis (SDS-PAGE), transfer to a fixed matrix or membrane and analysis by immunodetection using a specific antibody against the protein of interest. This method provides molecular mass information of individual proteins, and it can be used to distinguish between isoforms and post-translational modification states. There are many variations on the Western blot procedure that can be performed to improve detection, specificity, and quantitative power. In plants, Western blot has to be standardized to get a satisfactory signal and reproducible results, due to the complex nature of cell extracts. Here, I describe a protocol for a semi-quantitative Western blot using total maize protein and specific polyclonal antibodies raised against maize proteins of interest. The data are analyzed with ImageJ.
Keywords: Maize, Protein, Western blot, ImageJ
Materials and Reagents
Equipment
Software
Procedure
Data analysis
Notes
Recipes
Acknowledgments
MJAJ was supported by UC-MEXUS CONACYT and by NSF IOS-1238202 at Dr. Sarah Hake Lab (University of California Berkeley), where this protocol was developed. I would like to thank Dr. Sarah Hake and Dr. Madelaine Bartlett for their continuing scientific support, and to Dr. Ángel Gabriel Alpuche Solís and Alejandro Romo Avalos from IPICYT for their help in the preparation of figures and videos. I declare no conflict of interest regarding the implementation of this protocol.
References
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