Recombinant Anti-HIRA/HIR antibody [EPR7416] (ab129169)

We recommend to use 1%SDS Hot lysis prepare method.

We are unsure how to define the extra bands.

Intracellular Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling HIRA/HIR with purified ab129169 at 1/100 dilution (10µg/mL) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488, ab150077) secondary antibody was used at 1/2000. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue). 

Immunocytochemistry/ Immunofluorescence analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling HIRA/HIR with purified ab129169 at 1/200 dilution (5 µg/mL). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1/200 (2.5 µg/mL). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/1000 (2 µg/mL) dilution. DAPI (blue) was used as nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

ab129169 (Purified) at 1/50 dilution (2ug) immunoprecipitating HIRA/HIR in HEK-293 whole cell lysate.
Lane 1 (input): HEK-293 (Human embryonic kidney epithelial cell) whole cell lysate 10ug
Lane 2 (+): ab129169 & HEK-293 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab129169 in HEK-293 whole cell lysate
For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used at 1/1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST.

This antibody works in the lysates prepared with 1%SDS Hot lysis method in WB. No target band in input lane is due to the lysates prepared with RIPA method.

Overlay histogram showing HEK293 cells stained with unpurified ab129169 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab129169, 1/1000 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (0.1µg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HEK293 cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions. 

ab129169 (unpurified) at 1/50 dilution, staining HIRA/HIR in HeLa cells by Immunofluorescence.