Hrp labeled anti c terminal his tag antibody

Prediction of glycosylation sites in AamIGFBP-rP1 sequence was performed using online NetNGlyc 1.0 and NetOGlyc 4.0 servers (Blom et al., 2004 (link); Steentoft et al., 2013 (link)). Approximately 1 µg of affinity purified recombinant protein was treated with PNGaseF (New England BioLabs, Ipswich, MA, USA) or Enzyme Deglycosylation Mix (New England BioLabs) according to manufacturer’s protocols. Treated samples accompanied with non-treated controls were run on 12.5% SDS-PAGE and visualized by silver staining or western blot analysis using HRP-labeled anti C-terminal His-tag antibody (Life Technologies) and Metal Enhanced DAB Substrate Kit (Thermo Scientific).

Comparative analysis of insect cell-expressed rAamIGFBP-rP1 and human recombinant IGFBP-rP1 (Fitzgerald, Acton, MA, USA) trypsin digestion was performed. Around 0.5 µg of protein in 20 µL of PBS was subjected to digestion with bovine trypsin in the final concentrations of 5–100 µg/mL for 15 min at 37°C. Products of digestion were separated on 12.5% SDS-PAGE and visualized with standard Coomassie brilliant blue staining. In addition, western blot analysis was performed with digested samples using HRP-labeled anti C-terminal His-tag antibody (Life Technologies), as well as previously generated antibodies to A. americanum tick saliva proteins (Mulenga et al., 2013b (link)), to detect separated fragments. Positive signal was detected by using BioFX chemiluminescent reagent (SurModics).