| Products/Services Used | Details | Operation |
|---|---|---|
| Gene Synthesis | 1. The proteins were then transferred from the gel onto the PVDF membranes by eBlot L1 (GeneScript), then blocked with 5% skim milk to prevent non-specific binding and probed using anti-USP18 antibody to detect the protein of interest (4℃, overnight), and followed by an incubation with a secondary antibody (room temperature, 1h). 2. Protein Constructs ISG15: Full-length human ISG15 was codon-optimized and synthesized by Genscript (Nanjing) with an 8X Histag at N terminus, then cloned into a pET28a vector | Get A Quote |
| Proteins, Expression, Isolation and Analysis | Get A Quote |
ISG15 is a ubiquitin-like (Ubl) protein attached to substrate proteins by ISG15 conjugating enzymes whose dysregulation is implicated in a multitude of disease processes, but the probing of these enzymes remains to be accomplished. Here, we describe the development of a new activity-based probe ISG15-Dha (dehydroalanine) through protein semi-synthesis. In vitro cross-linking and cell lysate proteomic profiling experiments showed that this probe can sequentially capture ISG15 conjugating enzymes including E1 enzyme UBA7, E2 enzyme UBE2L6, E3 enzyme HERC5, the previously known ISG15 deconjugating enzyme (USP18), as well as some other enzymes (USP5 and USP14) which we additionally confirmed to impart deISGylation ... More
