Figure 5. Presence of Neu5Ac and Neu5,9Ac₂ in spectrin_VL by biochemical methods.

A. Enhanced sialylation demonstrated by IEF. Equal amounts (3.0 µg) of purified spectrin_VL, 60 kDa band and spectrin_N before and after removal of sialic acids were analyzed by IEF within a pH gradient of 3–10 and the respective bands visualized by silver staining. Lane M shows the pI markers. B–C. Enhanced sialylation in spectrin_VL. Equal amount (1.0 µg) of purified spectrin_VL and spectrin_N was analyzed by using DIG-glycan detection kits and total sialylation was compared based on the densitometric scores of spots (B). Representative bar graph of densitometric scores of corresponding spots (C). D–E. Detection of linkage-specific terminal sialic acids in spectrin_VL. Equal amount (2.0 µg) of spectrin_VL and spectrin_N was dot blotted on NC-paper and analyzed by DIG glycan and differentiation kit using SNA and MAA lectins following manufacturer's protocol (D). Densitometric scores of corresponding spots are shown as bar graph (E). F. Binding of ¹²⁵I-spectrin_VL/N with several sialic acid binding lectins. To demonstrate the presence or absence of terminal sialic acids, a fixed concentrations of ¹²⁵I-spectrin_VL/N were analyzed by binding with Sepharose/agarose bound WGA, SNA, MAA, Achatinin-H (25 µl bead volume) having specificity towards linkage specific sialic acids as described in materials and methods. Bound radioactivity of ¹²⁵I-spectrin_VL/N was measured by Gamma-counter and represented as bar graphs. G–H. Detection of sialylated tryptic fragments in spectrin_VL. The α and β subunits of purified spectrin_VL were digested separately by restricted amount of trypsin. Such controlled digested and extracted tryptic fragments were dried and redissolved and an aliquot was separated in SDS-PAGE (7.5%–15% gradient) (G). Subsequently the presence of sialic acids on resulting tryptic fragments was analyzed by binding with SNA-agarose and MAA-agarose separately and followed by electrophoresis on SDS-PAGE (7.5%–15% gradient) (H) as described in Materials and Methods. Lane M shows the molecular weight standerds.