Synthetic peptide (Human) corresponding to the C terminal peptide
Produced using Abcam's RabMAb® technology. RabMAb® technology is covered by the following U.S. Patents, No. 5, 675, 063 and/or 7, 429, 487.
This product is a recombinant rabbit monoclonal antibody.
Our Abpromise guarantee covers the use of ab32391 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|ICC/IF||1/100 - 1/500.
|WB||1/5000 - 1/10000. Predicted molecular weight: 46 kDa.|
|IHC-P||Use at an assay dependent concentration.|
ab172730 - Rabbit monoclonal IgG, is suitable for use as an isotype control with this antibody.
Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: GSK3B knockout HAP1 whole cell lysate (20 µg)
Lanes 1 - 2: Merged signal (red and green). Green - ab32391 observed at 46 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab32391 was shown to recognize GSK3B when GSK3B knockout samples were used, along with additional cross-reactive bands. 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 2000 dilution and 1/10000 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 1/10000 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.
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.
Image courtesy of an anonymous Abreview.