Figure 2. Conjugation of TMV to HA protein. Intact TMV 1295.10 was conjugated to HA protein in vitro as described, and analyzed on an SDS PAGE after staining with Coomassie Blue. (A) Glutaraldehyde, (B) EDC/Sulfo-NHS or (C) SMCC chemistries was successful in conjugating the HA protein to KLH (KLH-HA), TMV (TMV-HA) or as a self-conjugate (HA-HA). The mixture, as indicated by K+H or T+H, sampled before addition of the conjugation chemistry, shows an equimolar representation of both the proteins. The conjugate appears as a high molecular weight aggregate >190 kDa and in the gel stack. Successful conjugation was qualified by the complete absence of ‘free’ (unbound) HA protein as indicated. K-H (KLH-HA Conjugate), T-H (TMV-HA Conjugate), H-H (HA Self Conjugate).