Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human breast cancer tissue sections labeling RSK1 p90 with purified ab32114 at 1/100 dilution (15.4 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32114).

Immunocytochemistry/ Immunofluorescence analysis of K-562 (Human chronic myelogenous leukemia lymphoblast) cells labeling RSK1 p90 with purified ab32114 at 1:150 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) at 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.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32114).

Flow Cytometry analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling RSK1 p90 with purified ab32114 at 1/150 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32114).

ab32114 (purified) at 1/70 dilution (2ug) immunoprecipitating RSK1 p90 in K-562 whole cell lysate. K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate 10ug
Lane 2 (+): ab32114 & K-562 whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab32114 in K-562 whole cell lysate
For western blotting, VeriBlot for IP secondary antibody (HRP) (ab131366) was used at 1/1000 dilution.

Blocking and diluting buffer: 5% NFDM/TBST.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32114).

Overlay histogram showing HeLa cells stained with unpurified ab32114 (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 (ab32114, 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 HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32114).

Ab32114 (unpurified), at a 1/100 dilution, staining RSK1 p90 in paraffin embedded human colon tissue by immunohistochemistry.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab32114).