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

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : Rnf2 (RING2 / RING1B) knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 37 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.

Allele-1: 8 bp deletion in exon 2.

 

Allele-2: Insertion of the selection cassette in exon 2.