Nature research paper: Identification of trypsin-degrading commensals in the large intestine
JCM14589 strains were incubated with mouse recombinant PRSS2 for 20 min, washed with PBS and fixed with 4% paraformaldehyde-1% glutaraldehyde solution at room temperature for 2 h. After washing with 0.05 M PBS, the pellets were dehydrated in a graded series of ethanol . The dehydrated pellets were infiltrated with LRW resin . After infiltration, the samples were cured in gelatin capsules . Polymerized LRW blocks were sectioned using the Leica Ultracut UCT and 80 nm sections were obtained.
Nativ Marker Liquid Mix was used as the protein standard. For western blot analysis of blue native gels, proteins were blotted using the iBlot 2 Dry Blotting System with PVDF membranes . A list of the primers used for the generation of the recombinants is provided in Supplementary TableThe Pierce Fluorescent Protease Assay Kit was used to determine the protease activity of theculture supernatant, and recombinant 00502 and 00509 according to the manufacturer’s protocol.
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