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. Author manuscript; available in PMC: 2017 Aug 1.
Published in final edited form as: Methods. 2016 Mar 21;105:109–118. doi: 10.1016/j.ymeth.2016.03.009

Figure 5. SiMPull assay workflow and the step-by-step control experiments.

Figure 5

A) SiMPull assay workflow for studying the binding affinity and the diffusion activity of dsRBPs to dsRNA. B) Determination of TRBP concentration from lysate based on the calibration curve generated from Cy5 dye of known concentration. C) DsRBP specific pull-down test. TRBP-EYFP in cell lysates was pulled down to the imaging surface using biotinlyated GFP antibody, and the specific pull-down was tested using a pair of TRBP antibody and its interacting secondary antibody. D) RNA non-specific binding test to single-molecule imaging surface. DsRNA (pre-let-7) not ssRNA (polyU40) binds specifically to TRBP pulled-down surface while it show few non-specific binding to the imaging surface coated with biotinylated GFP antibody.