ab109230 stained HepG2 cells. The cells were 4%  formaldehdye fixed for 10 minutes at room temperature and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1hour at room temperature to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab109230 at 1/200) overnight at +4°C. The secondary antibody (pseudo-colored green) was ab96899 Dylight 488 goat anti-rabbit IgG used at a 1/1000 dilution for 1hour at room temperature. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1hour at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

All lanes : Anti-UBA52 antibody [EPR4547] (ab109230) at 1/1000 dilution

Lane 1 : Human placenta lysate
Lane 2 : Raji cell lysate
Lane 3 : Fetal kidney lysate
Lane 4 : 293T cell lysate
Lane 5 : Jurkat cell lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 15 kDa
Observed band size: 10 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of UBA52 in paraffin embedded Human brain tissue, using ab109230 at a 1/500 dilution.

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