Lane 1 & 3: Wild type HAP1 whole cell lysate (20 µg)
Lane 2 & 4: GSK3B knockout  HAP1 whole cell lysate (20 µg)

Lanes 1 - 4: Green - ab32391 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab32391 was shown to recognize GSK3B in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when GSK3B knockout samples were examined. Wild-type and GSK3B knockout samples were subjected to SDS-PAGE. Ab32391 and ab8245 (Mouse anti GAPDH loading control) were incubated overnight at 4°C at 1/2000 dilution and 1/10,000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

Immunohistochemical analysis of paraffin-embedded human breast tissue labelled with untreated ab75814 (phospho) (top-left) at a dilution of 1/1000, alkaline phosphatase treated ab75814 (phospho) (top-right) at a dilution of 1/1000, untreated ab32391 (bottom-left) at a dilution of 1/1000 and alkaline phophatase treated ab32391 (bottom-right) at a dilution of 1/1000. Ab97051 was used as secondary antibody at a dilution of 1/500 and counterstained with hematoxylin.

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling GSK3 beta with purified ab32391 at 1/500. Cells were fixed with 4% Paraformaldehyde and permeabilised with 0.1% tritonX-100. An Alexa Fluor® 488-conjugated goat anti-rabbit IgG (ab150077) at 1/1000 dilution was used as the secondary antibody. Cells were co-stained with ab7291, a mouse anti-tubulin antibody (1/1000) using ab150120, an Alexa Fluor® 594-conjugated goat anti-mouse IgG (1/1000) as the secondary antibody. Nuclei were counterstained with DAPI (blue).

For negative control 1, rabbit primary antibody was used followed by anti-mouse secondary antibody (ab150120).
For negative control 2, mouse primary antibody (ab7291) and anti-rabbit secondary antibody (ab150077) were used.

ELISA of GSK3 beta (phospho S9) peptide and GSK3 beta non-phospho peptide at 10 ng/ml. Detected with ab32391 at 0~1000 ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 was used as a secondary antibody.

ELISA of GSK3 beta (phospho S9) peptide and GSK3 beta non-phospho peptide at 1000 ng/ml. Detected with ab32391 at 0~1000 ng/ml. Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 was used as a secondary antibody.

Overlay histogram showing HeLa cells stained with ab32391 (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 (ab32391, 1/100 dilution) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.

Anti-GSK3 beta antibody [Y174] (ab32391) at 1/10000 dilution + A431 cell lysate.

Predicted band size: 46 kDa
Observed band size: 46 kDa

Immunohistochemical analysis of paraffin-embedded human breast cancer tissue labelled with untreated ab75814 (phospho) (top-left) at a dilution of 1/1000, alkaline phosphatase treated ab75814 (phospho) (top-right) at a dilution of 1/1000, untreated ab32391 (bottom-left) at a dilution of 1/1000 and alkaline phophatase treated ab32391 (bottom-right) at a dilution of 1/1000. Ab97051 was used as secondary antibody at a dilution of 1/500 and counterstained with hematoxylin.

IHC image of GSK3 staining in human breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6, epitope retrieval solution 1) for 20 minutes. The section was then incubated with ab32391, 1/200 diution, for 15 minutes at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

ICC/IF image of ab32391 stained DU145 cells. The cells were 4% formaldehyde fixed (10 minutes) and then incubated in 1 %BSA / 10 % normal goat serum / 0.3 M glycine in 0.1 % PBS-Tween for 1 hour to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab32391 at 1/200 dilution overnight at +4°C. The secondary antibody (green) was DyLight^® 488 goat anti- rabbit (ab96899) IgG (H+L) used at a 1/250 dilution for 1 hour. Alexa Fluor^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43 µM.

All lanes : Anti-GSK3 beta antibody [Y174] (ab32391) at 1/2500 dilution

Lane 1 : MCF7 (human breast adenocarcinoma cell line) cell lysate
Lane 2 : SW480 (human colorectal adenocarcinoma cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Donkey polyclonal IRDye 800CW at 1/15000 dilution

Predicted band size: 46 kDa
Observed band size: 46 kDa
Additional bands at: 37 kDa. We are unsure as to the identity of these extra bands.

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