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Functional Nano-Bioconjugates for Targeted Cellular Uptake and Specific Nanoparticle–Protein Interactions
Published in Grunwald Peter, Biocatalysis and Nanotechnology, 2017
Sanjay Mathur, Shaista Ilyas, Laura Wortmann, Jasleen Kaur, Isabel Gessner
To examine whether E-64@SPIONs can selectively capture cysteine proteases, a leaf extract of the model plant Arabidopsis thaliana with E-64@SPIONs and control beads (NPs without E-64) were incubated and the captured proteins were analyzed with an antibody against RD21, an abundant cysteine protease in Arabidopsis leaf extract. After antibody binding, the beads were washed and boiled in SDS sample buffer and eluted proteins were separated on a protein gel to analyze the protein blot using a secondary antibody that detects the RD21 antibody. The RD21 antibody was only detected using E-64@SPIONs and not by the control beads (Fig. 21.15). Pre-incubation of leaf proteomes with an excess of E-64 before adding the SPIONs prevented the capture of RD21 on the NPs, thereby demonstrating that capture was specific for cys proteases. E-64 contains reactive epoxy groups, which specifically conjugated with proteins of interest thereby giving us signals for the detection in western blots.
Atlantic Forest’s and Caatinga’s semiarid soils and their potential as a source for halothermotolerant actinomycetes and proteolytic enzymes
Published in Environmental Technology, 2023
Marghuel A. Vieira Silveira, Saara M. Batista dos Santos, Débora Noma Okamoto, Itamar Soares de Melo, Maria A. Juliano, Jair Ribeiro Chagas, Suzan P. Vasconcellos
The proteolytic activity was analysed using the following inhibitors: phenylmethylsulfonyl fluoride (PMSF), an inhibitor of serine proteases; ethylenediaminetetraacetic acid (EDTA), an inhibitor of metalloproteases, E-64, an inhibitor of cysteine protease and Pepstatin, an inhibitor of aspartic proteases, at a final concentration of 135 μM, 3, 1, 1 mM, respectively, in accordance with Dunn [41], with modifications. The effects of salt on hydrolytic activity were observed through the addition of NaCl to obtain a salt concentration in the range of 0–4M.