Isabelle Cristine da Costa, University of Manchester
Verified Customer
Applications: Western Blot
Species: Human
Sample: U2OS osteosarcoma cell line
Experimental Conditions: WB: Lysate prepared in RIPA buffer; 50 µg protein loaded; 4-8% Bis-Tris gel; PVDF membrane; wet transfer (150 mA, overnight, 4 °C); blocking in 1% BSA/TBST (30 min RT); primary antibody 1:500 O/N at 4 °C; washes in TBST (3×15 min); secondary antibody Invitrogen G-21234 (1:5000, 1 h RT); detection via ChemiDoc Bio-Rad with Amersham ECL.
Dilution: WB: 1:500
Results Summary: Antibody detects free NEDD8 (12 kDa) and NEDD8 conjugates; high specificity shown by reduction of conjugates with UBA3 inhibitor (3 µM, 1 h).
Comments: Highly specific and effective in detecting NEDD8 and conjugates in U2OS cells; clear reduction upon NEDDylation inhibition.
