Anti-Ubiquitin antibody [Ubi-1] (ab7254)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Ubiquitin antibody [Ubi-1] (ab7254)

Human normal placenta. Staining is localised in the cytoplasm and in the nuclei. Left panel: with primary antibody at 1 ug/ml. Right panel: isotype control. Sections were stained using an automated system DAKO Autostainer Plus, at room temperature: sections were rehydrated and antigen retrieved with the Dako 3 in 1 AR buffers citrate pH6.1 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 mins. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 min and detected with Dako envision flex amplification kit for mouse for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin antibody [Ubi-1] (ab7254)

ab7254 staining Ubiquitin in murine myocytes by Immunocytochemistry/ Immunofluorescence.

Isolated myocytes were treated with 10µM FCCP for 1 hour, or untreated as the control. Cells were then fixed in formaldehyde, permeabilized using 1% Triton X-100, then blocked with 10% horse serum for 1 hour at room temperature, incubated with ab7254 at a 1/200 dilution. The secondary used was an Alexa-Fluor 488 conjugated goat anti-mouse polyclonal, used at a 1/500 dilution.

Results show that with FCCP treatment, Ubiquitin is recruited onto mitochondria.

Image courtesy of an anonymous Abreview.

Western blot - Anti-Ubiquitin antibody [Ubi-1] (ab7254)

HeLa cells were co-transfected with a plasmid expressing a target protein together with Ubi expressing vector for 24 hours and either left untreated (Contr) or were treated with 10 µM MG-132 for 6 hours (+MG132). Then the protein of interest was pulled down using Flag agarose beads and and probed with ab7254 at a 1/2000 dilution. The secondary used was an Alexa-Fluor 680 conjugated goat anti-mouse polyclonal used at a 1/10000 dilution. The protein is known to be degraded through proteasome.

Image courtesy of an anonymous Abreview.