Fig. 10: P17 tag did not induce oligomerization of G12-scFv.
a Size-exclusion chromatography profiles of protein markers, G12-scFv-HA, and G12-scFv-HA-P17 proteins. Two hundred μl protein markers, G12-scFv-HA, and G12-scFv-HA-P17 proteins were loaded onto a Superdex 200 Increase 10/300 column using an ÄKTA purifier system. Proteins eluting by PBS at a flow rate of 0.5 ml/min were monitored by ultraviolet absorbance at 280 nm wavelength (A280nm). Elution peaks of protein markers (in black), including bovine thyroglobulin, bovine γ-globulin, chicken egg albumin, ribonuclease A, and p-aminobenzoic acid were labeled with corresponding molecular weights (MWs, 670 kDa, 150 kDa, 44.3 kDa, 13.7 kDa, and 0.137 kDa) in the left panel. Elution peaks of G12-scFv-HA (in green) and G12-scFv-HA-P17 (in blue) proteins were labeled with the MWs calculated from the calibration curve and equation in the right panel. Right panel: calibration curve and equation of average distribution constants (Kav) of protein markers and logarithms of their MWs (log (MW)). b Western blotting results of G12-scFv-HA and G12-scFv-HA-P17 proteins treated with formaldehyde or not. Five hundred ng G12-scFv-HA and G12-scFv-HA-P17 proteins were treated with 3% formaldehyde at 37 °C for 10 min or not, followed by the addition of 5×SDS sample loading buffer, and were detected by western blotting using anti-His mAb following separation by SDS-10% PAGE. The result of one representative experiment was shown. −, without formaldehyde cross-linking; +, with formaldehyde cross-linking.
