An electrophoretic mobility shift assay using the protein isolated from host plants.

Area of Science:

• Molecular Biology
• Plant Biotechnology

Background:

• Electrophoretic mobility shift assay (EMSA) is standard for DNA-protein interaction studies.
• Prokaryotic protein expression for EMSA often results in unnatural folding and lack of post-translational modifications, potentially affecting DNA binding accuracy.

Purpose of the Study:

• To develop a novel method for isolating and analyzing proteins from host plants for DNA-protein interaction studies.
• To overcome limitations of prokaryotic protein expression in EMSA.

Main Methods:

• Developed the protein from plants fluorescent EMSA (PPF-EMSA) method.
• Utilized transient gene transformation in plants for protein synthesis.
• Employed immunoprecipitation for protein isolation and cyanine 3 (Cy3) for DNA probe labeling.
• Performed fluorescent EMSA and super-shift fluorescent EMSA.

Main Results:

• Successfully isolated naturally folded, post-translationally modified proteins from *Betula platyphylla*, *Populus davidiana×P. bolleana*, and *Arabidopsis thaliana*.
• Demonstrated the utility of plant-derived proteins in fluorescent EMSA and super-shift fluorescent EMSA for accurate DNA binding analysis.
• PPF-EMSA enables true binding analysis due to proteins being in a natural state.

Conclusions:

• PPF-EMSA offers a convenient and rapid method for isolating proteins directly from host plants.
• The technique is not dependent on stable transformation, suggesting broad applicability for protein analysis.