Abstract
Protein immunoprecipitation (IP) is a method used to identify cellular protein complexes. Whence, antibodies that specifically recognize a native bait protein or an epitope tag fused to the protein of interest allow the protein of interest to be purified in complexes with other proteins. The extraction buffer must be stringent enough to dissociate weakly-interacting proteins to have a manageable number of proteins for mass spectrometry as well as to increase reproducibility between replicates, but not so stringent than can dissociate all the interactions. So, it is critical to have replicates to can identify reliable candidates to be interacting. As a model plant for grasses, it is important to have reproducible methods to analyze in vivo protein-protein interactions in maize. Here, I describe a simple and reproducible method for immunoprecipitation of native maize proteins using specific antibodies. This method also has been tested–with excellent results–for IP using magnetic beads coupled to an Anti-GFP antibody. I show the IP of a very low abundant membrane-localized protein called NOD (NARROW ODD DWARF).
Keywords: Maize, Protein purification, Immunoprecipitation, Western blot, Mass spectrometry
Materials and Reagents
Equipment
Procedure
Notes
Recipes
All of these buffers (Recipes 1-4) are suitable for immunoprecipitation of protein complexes. In general, milder buffer conditions will not dissociate protein-protein interactions. When a protein extract will be analyzed for phosphorylated proteins, add PhosSTOP phosphatase inhibitor mix (1 tablet for 10 ml of final buffer volume). Note: It is important to prepare all the solutions using MilliQ water, new protease-free tubes and filter the final stock solution through a 0.2 μm filter.
Acknowledgments
MJAJ was supported by UC-MEXUS CONACYT and by NSF IOS-1238202 at the Dr. Sarah Hake Lab (University of California Berkeley) and by NSF IOS-1652380 at the Dr. Madelaine Bartlett Lab (University of Massachusetts Amherst), where this protocol was developed. I would like to thank Dr. Sarah Hake and Dr. Madelaine Bartlett for their continuing scientific support. I declare no conflict of interest regarding the implementation of this protocol.
References
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