SuB3 a Simple Subcellular Fractionation Protocol for Localization Studies of Nucleic Acids and Proteins

Understanding the subcellular localization of RNA and proteins is critical to dissecting gene regulation in eukaryotic organisms. However, this task is elusive as existing fractionation methods often rely on protoplast isolation or commercial kits, that are labor-intensive, costly, and can introduce stress-induced transcriptomic and proteomic changes. Here, we present a simple, rapid, and cost-effective protocol for the fractionation of nuclear and cytoplasmic components directly from diverse plant tissues, that does not require protoplastization. This Sub cellular-fractionation protocol in 3 steps (a.k.a. “ bueno, bonito y barato” - spanish for “good, nice and cheap”-), referred to as “SuB3”, yields nuclear- and cytoplasmic- enriched subcellular fractions suitable for downstream applications such as RT-PCR, RNA/cDNA sequencing, and Western blotting. The procedure is based on sequential detergent-assisted extraction and centrifugation and enables the simultaneous isolation of RNA and protein from the same biological material. Due to its simplicity, speed, and broad compatibility, this protocol is a valuable tool for plant molecular biology laboratories investigating subcellular dynamics of gene expression.