Lane 1: Wild-type HAP1 cell lysate (20 µg)
Lane 2: S6K1 knockout HAP1 cell lysate (20 µg)
Lane 3: MCF7 cell lysate (20 µg)
Lane 4: HEK293 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab32529 observed at 68 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32529 was shown to recognize S6K1 when S6K1 knockout samples were used, along with additional cross-reactive bands. Wild-type and S6K1 knockout samples were subjected to SDS-PAGE. ab32529 and ab8245 (loading control to GAPDH) were diluted 1/5000 and 1/10000 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 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes : Anti-S6K1 antibody [E343] (ab32529) at 0.004 µg/ml (purified)

Lane 1 : Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate
Lane 2 : Mouse cerebellum lysate
Lane 3 : C6 (Rat glial tumor glial cell) whole cell lysate
Lane 4 : Rat cerebellum

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 59 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking and diluting buffer used: 5% NFDM/TBST.

Immunohistochemical analysis of Human breast cancer tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of C6 cells (Rat glial tumor glial cell)  labelling S6K1 with ab32529 at a dilution of 1:200, 11.1 µg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1:1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained with 1:200, 2.5μg/ml with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.

Immunohistochemical analysis of mouse testis tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

Immunohistochemical analysis of rat brain tissue labeling S6K1 with ab32529 at 1/500 dilution (4.4 μg/mL). The secondary antibody used was ImmunoHistoProbe one step HRP Polymer (ready to use). Secondary antibody only control-PBS instead of the primary antibody. Antigen retrieval was heat mediated using ab93684 (Tris/EDTA buffer, pH 9.0). The tissue was counterstained with Hematoxylin.

Flow cytometry analysis of Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling with ab32529 (purified) at 1/2200 dilution (1 µg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

Flow cytometry analysis of 293T (Human embryonic kidney epithelial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1 µg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

Flow cytometry analysis of C6 (Rat glial tumor glial cell) cells labelling with ab32529 (purified) at 1/2200 dilution (1 µg/mL) (red). Cells were fixed with 4% paraformaldehyde . Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) was used as the secondary antibody at 1/2000 dilution. Isotype control - 90% methanol . Unlabeled control - Rabbit monoclonal IgG (ab172730) / Black.

Lane 1: Neuro2a (Mouse neuroblastoma neuroblast) whole cell lysate, 10µg
Lane 2: Neuro2a whole cell lysate 350µg and ab32529, 2µg
Lane 3: Neuro2a cell lysate, 350µg and rabbit IgG (ab172730), 2µg

Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1/110 dilution (20 μg/ml). For western blotting, ab32529 at 1/500 and VeriBlot for IP (HRP) ab131366 was used for detection at 1/1000 dilution.

Blocking and diluting buffer used: 5% NFDM/TBST.

Lane 1: HEK293T (Human embryonic kidney epithelial cell) whole cell lysate, 10µg
Lane 2: HEK293T whole cell lysate, 10µg and ab32529, 2µg
Lane 3: HEK293T cell lysate, 350µg and rabbit IgG (ab172730) , 2µg

Purified ab32529 immunoprecipitating S6K1 in HEK293T cell lysates. Primary antibody was used at a 1/110 dilution (20 μg/ml). For western blotting,  ab32529 at 1/500 and VeriBlot for IP (HRP) ab131366 was used for detection at 1/1000 dilution.

Blocking and diluting buffer used: 5% NFDM/TBST.

Immunocytochemistry/Immunofluorescence analysis of NIH/3T3 (Mouse embryonic fibroblast) labelling with ab32529 at a dilution of 1:200, 11.1 μg/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A 1:1000 dilution (2μg/ml) was used for the secondary antibody Goat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained at 1:200 dilution, 2.5μg/ml with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594). Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.

Immunocytochemistry/Immunofluorescence analysis of MCF 7 (Human breast adenocarcinoma epithelial cell) labeling S6K1 with ab32529 at a dilution of 1:200, 11.1 ug/ml. Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. A dilution of 1/1000 (2μg/ml)  was used for the secondary antibodyGoat anti rabbit IgG (Alexa Fluor® 488, ab150077). The cells were co-stained at 1:200 dilution, 2.5μg/ml with Ab195889  Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) . Nuclei counterstained with DAPI (blue). Control: 1:1000 dilution.

Overlay histogram showing HeLa cells stained with ab32529 (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 (ab32529, 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.

Anti-S6K1 antibody [E343] (ab32529) at 1/10000 dilution + 293T cell lysate

Predicted band size: 59 kDa
Observed band size: 70 kDa why is the actual band size different from the predicted?