Ubiquitinated proteins were effectively isolated by Vx3(A7) protein. (A) A schematic diagram of Ubiquitinated proteins purification by binding with His-Vx3(A7)-eGFP protein conjugated with Ni-NTA agarose beads. (B) Ni-NTA agarose beads were used to purify Vx3(A7) protein, bacterial cell lysate, flowing fraction, washing fraction and elution fraction were collected and observed. (C) 12.5% SDS-PAGE was performed to detect the whole bacterial cell lysate proteins (lane 1) and the His-Vx3(A7)-eGFP fusion proteins (lane 2) which purified using Ni-NTA agarose Beads. (D) EL4 and B16-F10 tumor cells were treated with bortezomib (200 nM) and NH4Cl (10 mM). Whole cell lysates were prepared using tis-HCl lysis buffer. Ni-NTA agarose beads conjugated with Vx3(A7) proteins were used to isolate ubiquitinated proteins. Twenty micrograms of whole cell lysate (EL4, lane 2) and (B16-F10, lane 6), unbound (lane 3,7), washed (lane 4,8) and eluted fractions (lane 5,9) were loaded on SDS PAGE gel. (E, F) Western Blot analysis for ubiquitin proteins and K63 proteins in lysates (lane 1), unbound (lane 2), washed (lane 3) and eluted (lane 4) fractions harvested from EL4 (E) and B16-F10 (F) tumor cells using anti-ubiquitin and anti-K63 monoclonal antibodies, respectively.