All lanes : Anti-RING2 / RING1B / RNF2 antibody [EPR12245] (ab181140) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : RNF2 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 38 kDa
Observed band size: 42 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab181140 observed at 42 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

 ab181140 was shown to react with RING2 / RING1B / RNF2 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264845 (knockout cell lysate ab257640) lane below 42kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and RNF2 knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab181140 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human placenta tissue labeling RING2 / RING1B / RNF2 with ab181140 at 1/500 dilution followed by pre-diluted HRP-conjugated secondary antibody and counter-stained with Hematoxylin.

Perform heat mediated antigen retrieval with EDTA buffer pH 9 before commencing with IHC staining protocol.

Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: RING2 / RING1B / RNF2 knockout HAP1 cell lysate (20 µg)
Lane 3: HeLa cell lysate (20 µg)
Lane 4: Jurkat cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab181140 observed at 42 kDa. Red - loading control, ab7291, observed at 52 kDa.

ab181140 was shown to recognize RING2 / RING1B / RNF2 when RING2 / RING1B / RNF2 knockout samples were used, along with additional cross-reactive bands. Wild-type and RING2 / RING1B / RNF2 knockout samples were subjected to SDS-PAGE. ab181140 at a dilution of 1/1000 and ab7291 (loading control to alpha Tubulin) at a dilution of 1/10,000 were incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10,000 dilution for 1 h at room temperature before imaging.

Flow Cytometry analysis of HepG2 (human hepatocellular carcinoma) cells labeling RING2 / RING1B / RNF2 with purified ab181140 at 1/70 dilution (10ug/ml) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluor^® 488)(1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.

All lanes : Anti-RING2 / RING1B / RNF2 antibody [EPR12245] (ab181140) at 1/10000 dilution

Lane 1 : HeLa cell lysate
Lane 2 : 293 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugate at 1/1000 dilution

Predicted band size: 38 kDa

Immunofluorescent analysis of HepG2 cells (paraformaldehyde-fixed, 4%) labeling RING2 / RING1B / RNF2 with ab181140 at 1/250 dilution followed by Goat anti rabbit IgG (Alexa Fluor® 555) secondary at 1/200 dilution and counter-stained with DAPI (blue).