protein mixture standard  (Bio-Rad)

Journal: The Journal of Biological Chemistry

Article Title: Profiling the pancreatic cancer secretome with metabolic glycoengineering

doi: 10.1016/j.jbc.2026.111243

Figure Lengend Snippet: Far-Western blot analysis of the 1,3,4-O-Bu 3 ManNAz-enriched proteome in Capan-2 and hTERT-HPNE cells . ( A ) Capan-2 or ( B ) hTERT-HPNE samples were either untreated control cells (−) or cells incubated with 200 μM 1,3,4-O-Bu 3 ManNAz (+) for 8 to 96 h. Secreted proteins were precipitated from FBS-free cell culture supernatant and divided into aliquots that were subject to no glycosidase, PNGase F, or Protein Deglycosylation Mix II. Each sample was then separated by PAGE under identical electrophoretic conditions. The gels were first stained with SYPRO Protein Gel Stain to confirm equivalent protein loading and then subjected to far-Western analysis by biotinylating the azido-tagged glycoproteins using click chemistry conjugation with DBCO–PEG4–biotin, running the proteins on an SDS-PAGE gel, electroblotting them onto a nitrocellulose membrane, and probing for biotinylated proteins with streptavidin-conjugated horseradish peroxidase. FBS, fetal bovine serum; hTERT HPNE, human telomerase reverse transcriptase–immortalized human pancreatic nestin-expressing cell line.

Article Snippet: Protein samples were divided into three groups: a control group with no glycosidase treatment, PNGase F (New England Biolabs [NEB], P0705S) treated, and Protein Deglycosylation Mixture II (NEB, P6044S)–treated samples following the manufacturer’s instructions.

Techniques: Far Western Blot, Control, Incubation, Cell Culture, Staining, Western Blot, Conjugation Assay, SDS Page, Membrane, Reverse Transcription, Expressing