TY  - JOUR
TI  - Ratiometric Measurement of Protein Abundance after Transient Expression of a Transgene in Nicotiana benthamiana
AU  - Khosla, Aashima 
AU  - Nelson, David C.
VL  - 10
IS  - 17
PY  - 2020
DA  - 2020/09/05
SP  - e3747
C1  - Bio-protocol 2020;10:e3747
DO  - 10.21769/BioProtoc.3747
UR  - https://doi.org/10.21769/BioProtoc.3747
AB  - Ratiometric reporters are tools to dynamically measure the relative abundance of a protein of interest. In these systems, a target protein fused to a fluorescent or bioluminescent reporter is expressed with fixed stoichiometry to a reference protein fused to a second reporter. Both fusion proteins are encoded on a single transcript but are separated during translation by a 2A “self-cleaving” peptide. This approach enables changes in the relative abundance of a target protein to be detected sensitively, reducing variability in expression of the ratiometric reporter transgene that may occur across different tissues or transformation events. We recently developed a set of Gateway-compatible plant transformation vectors termed pRATIO that combine a variety of promoters, fluorescent and bioluminescent reporters, and 2A peptides derived from foot-and-mouth disease virus. Here, we describe in detail how to use the dual-fluorescent ratiometric reporter pRATIO3212 to examine the relative abundance of a target protein after transient expression in Nicotiana benthamiana leaves. For this example, we analyze degradation of the SUPPRESSOR OF MAX2 1 (SMAX1) protein from Arabidopsis thaliana in response to treatments with karrikins and rac-GR24. This protocol provides a simple, rapid, and readily scalable method for in vivo analysis of relative protein abundance in Agrobacterium-infiltrated Nicotiana leaf tissues.
KW  - Ratiometric reporter
KW  - Nicotiana benthamiana
KW  - Transient expression
KW  - 2A peptide
KW  - Fluorescence
KW  - Protein quantitation
JF  - Bio-protocol
SN  - 2331-8325
PB  - Bio-protocol LLC.
BIO101  - False