All lanes : Anti-UBE2I / UBC9 antibody [EP2938Y] (ab75854) at 1/10000 dilution

Lane 1 : U937 cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HUVEC cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat anti-rabbit HRP at 1/1000 dilution

Predicted band size: 18 kDa
Observed band size: 18 kDa

ab75854, at 1/100 dilution, staining UBE2I / UBC9 in human brain, by Immunohistochemistry using formalin-fixed, paraffin-embedded tissue.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (human T cell leukemia T lymphocyte) cells labeling UBE2I / UBC9 with purified ab75854 at 1/100 dilution (10 µg/mL). Cells were fixed in 100% Methanol. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL) was used as the secondary antibody only control.

UBE2I / UBC9 was immunoprecipitated using 0.5mg Jurkat whole cell extract, 5µg of Rabbit polyclonal to UBE2I / UBC9 and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Jurkat whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab75854.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 18kDa: UBE2I / UBC9; non specific - 60kDa: We are unsure as to the identity of this extra band.

Overlay histogram showing HepG2 cells stained with ab75854 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab75854, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.