All lanes : Anti-TRIM24 antibody [EPR22825-2] (ab256491) at 1/1000 dilution

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : TRIM24 knockout HeLa cell lysate
Lane 3 : Hap1 cell lysate
Lane 4 : A549 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 116 kDa
Observed band size: 140 kDa why is the actual band size different from the predicted?

Lanes 1- 2: Merged signal (red and green). Green - ab256491 observed at 140 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (ab8245) observed at 37 kDa.

 ab256491 was shown to react with Tripartite Motif Containing 24 in wild-type HeLa cells in western blot. The band observed in knockout cell line ab264963 (knockout cell lysate ab258246) lane below 140kDa may represent truncated forms and cleaved fragments. This has not been investigated further. Wild-type HeLa and TRIM24 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. ab256491 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) 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: 2 bp insertion in exon 1.

 

Allele-2: Insertion of the selection cassette in exon 1.