Figure 5. Exemplary results from dual color labeling experiment.

Proteoliposomes are separated in samples I, II, and III, and, after SNAP-labeling, in-gel fluorescence is measured in both channels. Subsequently, colloidal Coomassie staining is performed for normalization on total protein amount. All samples are prepared and loaded in duplicates. Sample I is labeled with the membrane-impermeable SNAP-Cell Alexa488. Sample II is labeled with the membrane-permeable SNAP-Cell 647-SIR and served as 100% protein signal. Sample III is blocked on the outside with membrane-impermeable SNAP-Surface Alexa488 first, followed by the labeling of inward-facing proteins only with the membrane-permeable SNAP-Cell 647-SIR. All fluorescence intensities are normalized on corresponding Coomassie signal. The normalized signal intensities in the SNAP-Cell 647-SIR channel are used to determine the percentage of inside-oriented protein by division of the signals derived from sample III (only inside labeling) by sample II (all protein labeled), showing here 25%. Bleed-through in the SNAP-Cell 647-SIR channel can be determined by division of the signals derived from sample I (no label) by sample II (all protein labeled), showing here less than 1%.