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Molecular characterization of the WGA-Fc chimera

SL Susie Coutinho Liedke
DM Daniel Zamith Miranda
KG Kamilla Xavier Gomes
JG Jorge Luis S. Gonçalves
SF Susana Frases
JN Joshua D. Nosanchuk
MR Marcio L. Rodrigues
LN Leonardo Nimrichter
JP José Mauro Peralta
AG Allan J. Guimarães
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To evaluate protein structure, aliquots of WGA-Fc were diluted in denaturing (12.5 mM Tris pH 6.8, 4% glycerol, 0.4% SDS, 1% ß-mercaptoethanol, 0.005% w/v bromophenol blue, dH2O) and non-denaturing (as above, but without 1% ß-mercaptoethanol) buffers and incubated at 95 °C for 5 min. An IgG2a mAb 12D3 against H. capsulatum heat-shock protein 60 KDa was used as a control16. The proteins were resolved by SDS-PAGE, transferred to nitrocellulose membrane and blocked with 5% milk solution in 0.1% TBS-T overnight. For immunodetection of chimeras or control antibodies, a goat anti-mouse IgG conjugated with alkaline–phosphatase was used and after washes with TBS-T, the membrane was developed with 5-bromo-4chloro-3-indolyl phosphate/NBT reagent (Sigma-Aldrich).

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