Anti-Ikaros antibody [EPR13791] - C-terminal

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Ikaros antibody [EPR13791] - C-terminal (AB190691)

Intracellular Flow Cytometry analysis of Jurkat (human acute T cell leukemia) labelling Ikaros with purified ab190691 at 1/60 (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Alexa Fluor^® 488 goat anti-rabbit IgG (1/2000) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Ikaros antibody [EPR13791] - C-terminal (AB190691)

Immunofluorescent analysis of 4% paraformaldehyde fixed Jurkat cells labeling Ikaros with ab190691 at 1/250 followed by Goat anti rabbit IgG (Alexa Fluor® 555) at 1/200 and conterstained with DAPI.

• IP

Lab

Immunoprecipitation - Anti-Ikaros antibody [EPR13791] - C-terminal (AB190691)

50-70 kDa Ikaros was immunoprecipitated from 0.35mg Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab190691 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab190691 at 1/1000 dilution (0.71 μg/ml). VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/5000 dilution. Lane 1 (Input) : Ramos (human Burkitt's lymphoma B lymphocyte) whole cell lysate 10 μg. Lane 2 (+) : Ramos whole cell lysate. Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab190691 in Ramos whole cell lysate. Blocking buffer and concentration : 5% NFDM/TBST. Diluting buffer and concentration : 5% NFDM/TBST. Exposure time : 15 seconds.

All lanes:

Immunoprecipitation - Anti-Ikaros antibody [EPR13791] - C-terminal (ab190691) at 1/30 dilution

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

Predicted band size: 57 kDa

Observed band size: 50-70 kDa

false

Exposure time: 15s

• WB

Lab

Western blot - Anti-Ikaros antibody [EPR13791] - C-terminal (AB190691)

Lanes 1 - 4 : Merged signal (red and green). Green - ab190691 observed at 50-70 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab190691 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 ab190691 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 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.

All lanes:

Western blot - Anti-Ikaros antibody [EPR13791] - C-terminal (ab190691) at 1/1000 dilution

Lane 1:

Wild-type Jurkat cell lysate at 20 µg

Lane 2:

IKZF1 knockout Jurkat cell lysate at 20 µg

Lane 3:

Daudi cell lysate at 20 µg

Lane 4:

HeLa cell lysate at 20 µg

Predicted band size: 57 kDa

Observed band size: 50-70 kDa

false

• WB

Supplier Data

Western blot - Anti-Ikaros antibody [EPR13791] - C-terminal (AB190691)

All lanes:

Western blot - Anti-Ikaros antibody [EPR13791] - C-terminal (ab190691) at 1/10000 dilution

Lane 1:

Raji cell line lysate at 20 µg

Lane 2:

Jurkat cell line lysate at 20 µg

Lane 3:

Ramos cell line lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated antibody at 1/1000 dilution

Predicted band size: 57 kDa

Observed band size: 50-70 kDa

false