重组Anti-HOXB4抗体[EP1919Y] (ab76093)

Immunocytochemistry/ Immunofluorescence analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling HOXB4 with purified ab76093 at 1:100 dilution (10 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.

ab76093 (Purified) at 1:60 dilution (2 µg) immunoprecipitating HOXB4 in K-562 whole cell lysate.
Lane 1 (input): K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10 µg
Lane 2 (+): ab76093 & K-562 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab76093 in K-562 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.

Intracellular Flow Cytometry analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling HOXB4 with purified ab76093 at 1/100 dilution (10 µg/ml) (Red). Cells were fixed with 4% Paraformaldehyde and permeabilised with 90% Methanol. A Goat anti rabbit IgG (Alexa Fluor^® 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).

 

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human lung carcinoma tissue sections labeling HOXB4 with purified ab76093 at 1:100 dilution (11.86 µg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). ImmunoHistoProbe one step HRP Polymer (ready to use) was used as the secondary antibody. Negative control: PBS instead of the primary antibody. Hematoxylin was used as a counterstain.

Anti-HOXB4 antibody [EP1919Y] (ab76093) at 1/1000 dilution (Purified) + K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysates at 15 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 28 kDa
Observed band size: 34 kDa why is the actual band size different from the predicted?

Immunohistochemical analysis of paraffin-embedded human placental tissue, using unpurified ab76093, at 1/100 dilution.

Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Overlay histogram showing K562 cells stained with unpurifiedab76093 (red line). The cells were fixed with 80% methanol (5 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 (ab76093, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.