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. 2016 Feb 8;7(13):15606–15617. doi: 10.18632/oncotarget.7244

Figure 1. IVIG was fractionated with SNA affinity chromatography.

Figure 1

A. Typical fractionation chromatogram of IVIG on a SNA agarose column. Three fractions including one SNA non-binding fraction named FT IVIG (elution with Binding Buffer) and two SNA binding fractions separately named E1 IVIG (elution with Elution Buffer 1) and E2 IVIG (elution with Elution Buffer 2) were obtained. B. IVIG fractions were separated with SDS-PAGE and stained with silver. Two higher molecular weight bands existed above the 50 kDa heavy chain and the 25 kDa light chain (arrow) respectively of E1 IVIG and E2 IVIG which fell back to the normal molecular weight following deglycosylation with PNGase F. C–E. The identities of these two extra bands were established with Western blot. The band above the 50 kDa heavy chain was Ig γ heavy chain (C), the band above the 25 kDa light chain was the mixture of kappa light chain (D) and lambda light chain (E). F&G. The presence and the linkage patterns of sialic acids on IVIG fractions were examined with Lectin blot. Sialic acids on IVIG fractions were linked through α-2,6 but not α-2,3, which were conformed with SNA (F) and MAA (G). Strong SNA positive signals existed on both the heavy chain and the light chain of E1 IVIG and E2 IVIG. Weak SNA positive signal existed on the heavy chain of FT IVIG (F). No MAA positive signal was detectable on any IVIG fractions (G). Fetuin and Asialofetuin were used as control glycoproteins.