Figure 5. Size exclusion chromatography (SEC) of the complex of TNF-alpha and the ubiquitin variant 10F.

(A) SEC in presence of detergent Tween-20. Samples of fluorescein-labeled 10F (10 µM), TNF-alpha (60 µM), and a mixture of both, were pre-incubated for 10 min at 4°C and loaded on a Superose 12 size-exclusion column. Experiments were performed with sample and running buffer, both containing 0.05% (v/v) Tween-20, and detection wavelengths of 280 nm (blue line) and 495 nm (red dashed line). Note that there is no significant shift of the TNF-alpha elution volume when comparing the elution profiles of TNF-alpha alone and the mixture. Fluorescein itself did not co-elute with TNF-alpha (data not shown). (B) SEC in absence of detergent Tween-20. For analysis of Tween-20 independent complex formation, a detergent-free mixture of unlabeled 10F (10 µM) and TNF-alpha (60 µM) was incubated at 4°C and aliquots were analyzed as described before using running buffer without Tween-20. Note that the area of the 10F peak (16.5 ml) decreases during prolonged incubation. Dashed lines mark the borders of the fraction used for SDS-PAGE analysis. (C) SDS-PAGE analysis of the detergent-free SEC complex fractions. Aliquots of appropriate SEC fractions were analyzed by SDS-PAGE (4–12% gradient gel) followed by Coomassie staining. M: Fermentas PageRuler Unstained, 1: control TNF-alpha (2 µg), 2: control 10F (2 µg), 3: 0.17 h preincubation, 4: 1 h, 5: 2 h*, 6: 6 h, 7: 12 h, 8: 24 h*, 9: 44 h. *To reduce complexity chromatograms were not included in (B).