重组Anti-RSK1 p90抗体[E4]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RSK1 p90 antibody [E4] (AB32114)

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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RSK1 p90 antibody [E4] (AB32114)

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

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-RSK1 p90 antibody [E4] (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.

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-RSK1 p90 antibody [E4] (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.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-RSK1 p90 antibody [E4] (AB32114)

ab32114 (unpurified), at a 1/100 dilution, staining RSK1 p90 in paraffin embedded human colon tissue by immunohistochemistry.

• IP

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Immunoprecipitation - Anti-RSK1 p90 antibody [E4] (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.

All lanes:

Immunoprecipitation - Anti-RSK1 p90 antibody [E4] (ab32114)

Predicted band size: 82 kDa

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• WB

Lab

Western blot - Anti-RSK1 p90 antibody [E4] (AB32114)

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.

All lanes:

Western blot - Anti-RSK1 p90 antibody [E4] (ab32114)

Predicted band size: 82 kDa

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• WB

Lab

Western blot - Anti-RSK1 p90 antibody [E4] (AB32114)

Western blot : Anti-RSK1 p90 antibody [E4] ab32114 staining at 1/500 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 83 kDa in Wild-type MCF7 cell lysates with no signal observed at this size in RPS6KA1 knockout MCF7 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3pc Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-RSK1 p90 antibody [E4] (ab32114) at 1/500 dilution

Lane 1:

Wild-type MCF7 at 20 µg

Lane 2:

Western blot - Human RPS6KA1 knockout MCF7 cell line (ab289311) at 20 µg

Lane 3:

PC-3 at 20 µg

Lane 4:

Calu-3 at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 83 kDa

Observed band size: 83 kDa,37 kDa

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• WB

Lab

Western blot - Anti-RSK1 p90 antibody [E4] (AB32114)

All lanes:

Western blot - Anti-RSK1 p90 antibody [E4] (ab32114) at 1/1000 dilution

Lane 1:

MCF7 (Human breast adenocarcinoma epithelial cell) whole cell lysates at 20 µg

Lane 2:

K-562 (Human chronic myelogenous leukemia lymphoblast) whole cell lysate at 20 µg

Lane 3:

Mouse lung lysates at 20 µg

Lane 4:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 82 kDa

Observed band size: 83 kDa

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• WB

Unknown

Western blot - Anti-RSK1 p90 antibody [E4] (AB32114)

All lanes:

Western blot - Anti-RSK1 p90 antibody [E4] (ab32114) at 1/1000 dilution

All lanes:

MCF7 cell lysate at 10 µg

Predicted band size: 82 kDa

Observed band size: 90 kDa

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