All lanes : Anti-Ikaros antibody [EPR13790] (ab191394) at 1/10000 dilution

Lane 1 : Wild-type Jurkat cell lysate
Lane 2 : IKZF1 knockout Jurkat cell lysate
Lane 3 : Daudi cell lysate
Lane 4 : HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 58 kDa
Observed band size: 50-70 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab191394 observed at 50-70 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab191394 was shown to react with Ikaros in wild-type Jurkat cells in western blot with loss of signal observed in IKZF1 knockout sample. Wild-type and IKZF1 knockout Jurkat cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab191394 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 10000 dilution and a 1 in 20000 dilution respectively. Blots were incubated with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded Human thymus tissue labeling Ikaros with ab191394 at 1/1400 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.
Inset: Negative control: using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

All lanes : Anti-Ikaros antibody [EPR13790] (ab191394) at 1/10000 dilution

Lane 1 : Raji cell lysate
Lane 2 : Ramos cell lysate
Lane 3 : MOLT4 cell lysate

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 58 kDa

Based on the sequence analysis, ab191394 recognizes seven isoforms with the predicted MWs of 58KDa, 48KDa, 48KDa, 43KDa, 41KDa, 32KDa and 53KDa, respectively.

Immunohistochemical analysis of paraffin-embedded Mouse spleen tissue labeling Ikaros with ab191394 at 1/1400 dilution followed by pre-diluted HRP Polymer for Rabbit IgG secondary antibody and counter-stained with Hematoxylin.
Inset: Negative control: using PBS instead of primary antibody.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Flow Cytometric analysis of MOLT4 cells (paraformaldehyde-fixed, 2%) labeling Ikaros with ab191394 at 1/130 dilution (red) or a rabbit IgG (negative) (green), followed by Goat anti rabbit IgG (FITC) secondary at 1/150 dilution.