This data was developed using ab76117, the same antibody clone in a different buffer formulation.

Lane 1: Wild-type HAP1 cell lysate (40 µg)

Lane 2: RSK4 knockout HAP1 cell lysate (40 µg)

Lane 3: MCF7 cell lysate (20 µg)

Lane 4: HEK293 cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab76117 observed at 90 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab76117 was shown to recognize RSK4 when RSK4 knockout samples were used, along with additional cross-reactive bands. Wild-type and RSK4 knockout samples were subjected to SDS-PAGE. ab76117 and ab8245 (loading control to GAPDH) were diluted at 1/250 and 1/10,000 dilution 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 hour at room temperature before imaging.

All lanes : Anti-RSK4 antibody [EP1982Y] (ab76117) at 1/1000 dilution (purified)

Lane 1 : SH-SY5Y cell lysate
Lane 2 : Caco-2 cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : HRP goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 84 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

This data was developed using ab76117, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.

Immunofluorescence staining of HEK293 cells with purified ab76117 at a working dilution of 1/1000, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab76117 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.

Immunohistochemical staining of paraffin embedded rat kidney with purified ab76117 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.

Immunohistochemical staining of paraffin embedded mouse cerebral cortex with purified ab76117 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.Immunohistochemical staining of paraffin embedded human kidney with purified ab76117 at a working dilution of 1/250. The secondary antibody used is HRP goat anti-rabbit IgG H&L (ab97051) at 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0. PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.Overlay histogram showing Neuro-2a cells fixed in 2% PFA and stained with purified ab76117 at a dilution of 1 in 70 (red line). The secondary antibody used was FITC goat anti-rabbit at a dilution of 1 in 500. Rabbit monoclonal IgG was used as an isotype control (black line) and cells incubated in the absence of both primary and secondary antibody were used as a negative control (blue line).

Anti-RSK4 antibody [EP1982Y] (ab76117) at 1/500 dilution (unpurified) + SH-SY5Y cell lysate at 10 µg

Secondary
HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 84 kDa
Observed band size: 90 kDa why is the actual band size different from the predicted?

This data was developed using ab76117, the same antibody clone in a different buffer formulation.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.Unpurified ab76117, at 1/50 dilution, staining RSK4 in paraffin-embedded human brain glioma tissue. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

This data was developed using ab76117, the same antibody clone in a different buffer formulation.Immunofluorescent staining of SH-Sy5y cells using unpurified ab76117 at 1/100 dilution.