Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: RSK1 p90 knockout HAP1 cell lysate (20 µg)
Lane 3: MCF7 cell lysate (20 µg)
Lane 4: K562 cell lysate (20 µg)
Lanes 1 - 4: Merged signal (red and green). Green - ab32114 (unpurified) observed at 88 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32114 was shown to recognize RSK1 p90 when RSK1 p90 knockout samples were used, along with additional cross-reactive bands. Wild-type and RSK1 p90 knockout samples were subjected to SDS-PAGE. ab32114 and ab8245 (loading control to GAPDH) were diluted at 1/500 and 1/10 000 respectively and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10 000 dilution for 1 h at room temperature before imaging.

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 heat mediated antigen retrieval 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.

All lanes : Anti-RSK1 p90 antibody [E4] (ab32114) at 1/1000 dilution (Purified)

Lane 1 : MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate
Lane 3 : Mouse lung lysates
Lane 4 : PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 83 kDa
Observed band size: 83 kDa

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.

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).

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.

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

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.

Anti-RSK1 p90 antibody [E4] (ab32114) at 1/1000 dilution (unpurified) + MCF7 cell lysate at 10 µg

Predicted band size: 83 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?