All lanes : Anti-UBE2M/UBC12 antibody [EPR5333] (ab109507) at 1/10000 dilution

Lane 1 : HEK-293T cell lysate
Lane 2 : Ramos cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 21 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded human colonic adenocarcinoma tissue using ab109507 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

Immunocytochemistry/ Immunofluorescence analysis of Ramos (human Burkitt's lymphoma B lymphocyte) cells labeling UBE2M/UBC12 with purified ab109507 at 1/250 dilution (8 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. 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.

Overlay histogram showing Ramos cells stained with ab109507 (red line). The cells were fixed with 4% paraformaldehyde (10 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 (ab109507, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Immunohistochemical analysis of paraffin-embedded Human lung carcinoma tissue using ab109507 at a dilution of 1/100.

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.