Electrophoretic Mobility Shift Assay of in vitro Phosphorylated RNA Polymerase II Carboxyl-terminal Domain Substrates

Abstract

Eukaryotic RNA polymerase II transcribes all protein-coding mRNAs and is highly regulated. A key mechanism directing RNA polymerase II and facilitating the co-transcriptional processing of mRNAs is the phosphorylation of its highly repetitive carboxyl-terminal domain (CTD) of its largest subunit, RPB1, at specific residues. A variety of techniques exist to identify and quantify the degree of CTD phosphorylation, including phosphorylation-specific antibodies and mass spectrometry. Electrophoretic mobility shift assays (EMSAs) have been utilized since the discovery of CTD phosphorylation and continue to represent a simple, direct, and widely applicable approach for qualitatively monitoring CTD phosphorylation. We present a standardized method for EMSA analysis of recombinant GST-CTD substrates phosphorylated by a variety of CTD kinases. Strategies to analyze samples under both denatured/reduced and semi-native conditions are provided. This method represents a simple, direct, and reproducible means to monitor CTD phosphorylation in recombinant substrates utilizing equipment common to molecular biology labs and readily applicable to downstream analyses including immunoblotting and mass spectrometry.

Protocol for detecting lncRNA-protein interactions in vitro by tRSA RNA pull-down assay

“… Note: The effects of GST-SAD2 and GST proteins expressions are evaluated using 1 mL of both the uninduced samples (-IPTG) and induced samples (+IPTG), then detected by electrophoresis using an SDS-PAGE gel with an 5% stacking gel and a 12% resolving gel following by Coomassie staining with Coomassie brilliant blue staining solution. 4 Before running an SDS-PAGE gel, cells are resuspended with 120 μL 1× SDS loading buffer, and take 20 μL of resuspension that are denatured by heating at 100°C for 10 min for electrophoresis. If enrichments of GST-SAD2 and GST are observed in the induced samples but not in the uninduced samples, then GST-SAD2 and GST have been successfully expressed ( Figure 1 B).…”

What's all the phos about? Insights into the phosphorylation state of the RNA polymerase II C-terminal domainviamass spectrometry

“…Initial efforts to detect phosphorylation of the CTD utilized gel electrophoresis since hyperphosphorylation is known to change the mobility of the bands dramatically. 5,8 While this method has the advantage of being fast with no requirement of special equipment, no information on the specific site of phosphorylation can be derived. 9 Thus, site-specific CTD antibodies were generated and optimized, leading to exponential growth in the understanding of CTD PTMs.…”