Figure 2.

Coomassie-stained white light and Odyssey fluorescence images of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels. Each image row represents a single antibody. Conjugated samples of all 3 dyes both intact and reduced were run on the same gel both intact and after reduction by β-mercaptoethanol. PL represents the protein ladder, molecular weights for the ladder are depicted left of the coomassie stained bands. Intact antibodies showed clearly defined single bands around 150 kDa, in line with the unmodified reference antibody (ST) lane. Reduced gels showed 2 clearly defined bands, at 50 kDa for heavy chain and 25 kDa for light chain. Fluorescence shows comparable results for all intact antibodies, which show up as clear single bands in their respective fluorescent wavelength (Green: 800 nm, red: 700 nm). After reduction, fluorescent bands can be seen at the 50 kDa and 25 kDa locations for all antibodies, except when conjugated to ZW800. A poorly defined band can be observed at the very bottom of the gel for lanes of reduced ZW800 tracers, while negligible signal is detectable at the reduced band locations. Abbreviations: Fluo: Odyssey CLX fluorescence scan; PL: protein ladder; ST: unconjugated protein reference; 800CW: lane with antibody conjugated to IRDye 800CW; 680LT: lane with antibody conjugated to IRDye 680LT; ZW800: lane with antibody conjugated to ZW800-1.