cy5 affinipure donkey anti rabbit igg  (Jackson Immuno)

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Multivalent weak contacts shape chaperone-nascent protein interactions

doi: 10.1073/pnas.2607201123

Figure Lengend Snippet: A. Schematic of the experimental setup. Ribosome-nascent protein complexes (RNCs) are surface-immobilized via the messenger RNA (mRNA). TIRF illumination allows detection of Atto532-labeled trigger factor molecules upon RNC binding. The nascent protein is labeled with sulfo-Cy5-derivatized SpyCatcher (grey) at an N-terminal SpyTag. B. Time-integrated wide-field TIRF images showing the initial excitation (left; 10 frames at 638 nm) and combined excitation (right; 10 frames at 638 nm, 10 frames at 532 nm, 10 frames at 638 nm). Initial and final excitation at 638 nm is used to visualize the positions of individual immobilized RNCs (red channel on the left). Dots in the green channel (right) that co-localize with the red dots represent RNC-bound trigger factor molecules. C. Example trajectory of fluorescence emission intensity versus time at the position of an RNC135, which is detected by red illumination during the initial 10 frames. Subsequently, red illumination is turned off, and green illumination is turned on. Individual trigger factor binding and dissociation events are detected as spikes in the recorded fluorescence emission intensity. Frame rate: 10 Hz. D. Comparison of binding events detected with wild-type trigger factor (WT) and a mutant defective in ribosome binding (mut), demonstrating specific binding to surface-immobilized RNCs.

Article Snippet: SpyCatcher with a N-terminal cysteine ( 29 ) was labeled with maleimide-modified sulfo-Cy5 (Lumiprobe) similar to the procedure used for labeling trigger factor.

Techniques: Labeling, Binding Assay, Fluorescence, Comparison, Mutagenesis