electrophoretic mobility shift assays emsa biotin labeled probes  (Beyotime)

Journal: Frontiers in Plant Science

Article Title: Melatonin Alleviates Low-Temperature Stress via ABI5-Mediated Signals During Seed Germination in Rice ( Oryza sativa L.)

doi: 10.3389/fpls.2021.727596

Figure Lengend Snippet: Expression of CATALYSE 2 ( OsCAT2 ) under LT stress treated with exogenous MT and electrophoretic mobility shift assay (EMSA) assays for a direct interaction between OsABI5 and the OsCAT2 promoter. (A) Transcript abundance of OsCAT2 under LT stress (constant 20°C for 7 days) without (LT20) or with MT (LT20 + MT) treatment in WT and abi5-1 and abi5-2 mutants. (B) An EMSA validated the interaction of OsABI5 with the promoters of OsCAT2. The purified OsABI5 protein obtained using prokaryotic expression was used for the in vitro assay, and the EBNA protein was used as a negative control. Unlabeled probes (competitor) were subjected to cold competition experiments (100×). Different letters denote significant variations between the treatments, and the average values were measured by Tukey’s HSD test at p < 0.05. Data were represented as mean ± SE of six biological replicates. “+” and “–” indicate the presence or absence, respectively, of proteins and probes in the loading mixture. About 4% of the polyacrylamide gel was used here.

Article Snippet: In parallel, 25-bp DNA fragments from the OsCAT2 promoter [harboring the ABA response element (ABRE) motif, − 505th to − 481th distance to transcription start site (TSS)] were biotin-labeled at the 3′-end by using the electrophoretic mobility shift assay (EMSA) Probe Biotin Labeling kit (Promega, Madison, WI, United States).

Techniques: Expressing, Electrophoretic Mobility Shift Assay, Purification, In Vitro, Negative Control