Purity of SNAP-tagged IQGAP1 proteins and comparison of the labeled actins used in TIRF assays. (A) Table of predicted molecular weight and oligomeric state of untagged and SNAP-tagged IQGAP1 proteins. (B) SDS-PAGE gels of untagged and SNAP-tagged IQGAP1 proteins in A. (C) Images from TIRF reactions comparing the actin polymerization of different fluorescent labeling strategies. Reactions contain 1 µM actin labeled with either 10% Dylight 405, 10% Oregon Green (OG), 10% Alexa 488, or 10% Alexa 647 dyes. Dylight 405- and OG-actin were labeled on cysteine (Cys) 374, whereas 488- and 647-actin were labeled on accessible lysine (Lys) residues of polymerized actin. Scale, 10 µm. (D) Mean actin filament elongation rates from reactions in C (n = 17 filaments per reaction, 51 total per condition [dots]; from three different trials). Error bars, SD. Statistics, ANOVA: (a) P ≤ 0.05 compared with all other conditions; ns, P ≥ 0.05 compared with each condition under the line. Source data are available for this figure: SourceData FS2.