Anti-Ubiquitin Antibody (A85455)

Immunofluorescent analysis of HeLa cells by Anti-Ubiquitin Antibody. Proteasomal inhibition leads to formation of ubiquitin-positive cytoplasmic inclusions in HeLa cells. Cells grown on glass coverslips were maintained in normal medium (Ct) or treated with lactacystin (Lc) at 10µm for 24 hours, fixed, and then stained with Anti-Ubiquitin Antibody (red) and Anti-Vimentin Antibody (A85421 | green). The blue is DAPI staining of nuclear DNA. Note the diffuse ubiquitin staining in control cells throughout the cytoplasm and ubiquitin-positive well-defined cytoplasmic inclusions in lactacystin-treated cells.

Western blot analysis of HEK293 cell lysates using Anti-Ubiquitin Antibody (1:5000 | green) and Anti-Beta Tubulin Antibody (A85428 | 1:10000 | red | Loading Control). Cells were maintained in normal medium (Control | Ct) or treated with proteasome inhibitor lactacystin (Lc) at 10 µm for 16 h. Lysed cells were electrophoresed on 4-20% SDS-PAGE, and blotted to PVDF membrane. Note the smeary-patterned reactivities detected above the 200kDa standard that presumably represent accumulation of ubiquitinated proteins in proteasome inhibitor-Lc treated cells. Prominent band at 8kDa corresponds to monoubiquitin.

Western blot analysis of HEK293 cell lysates using Anti-Ubiquitin Antibody (A85455), at a dilution of 1:5,000, in green. The lanes contain samples of: [1] Protein standards, in red, [2] cells maintained in normal medium, and [3] cells treated with proteasome inhibitor lactacystin (Lc) at 10 µM for 16 hours. The lysate was subjected to electrophoresis on a 4-20% SDS-PAGE gel, then electrophoretically transferred to PVDF membranes. The smear detected above the 200 kDa standard represents accumulations of ubiquitinated proteins in the Lc treated cells. The prominent band at approximately 8 kDa corresponds to monoubiquitin. The same blot was simultaneously probed with Anti-beta Tubulin Antibody (A85428), at a dilution of 1:10,000, in red, used as a loading control.