The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use a concentration of 5 µg/ml.
Use a concentration of 1 µg/ml. Detects a band of approximately 47 kDa (predicted molecular weight: 47 kDa).
Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
Participates in the Wnt signaling pathway. Implicated in the hormonal control of several regulatory proteins including glycogen synthase, MYB and the transcription factor JUN. Phosphorylates JUN at sites proximal to its DNA-binding domain, thereby reducing its affinity for DNA. Phosphorylates MUC1 in breast cancer cells, and decreases the interaction of MUC1 with CTNNB1/beta-catenin. Phosphorylates CTNNB1/beta-catenin. Phosphorylates SNAI1. Plays an important role in ERBB2-dependent stabilization of microtubules at the cell cortex. Prevents the phosphorylation of APC and CLASP2, allowing its association with the cell membrane. In turn, membrane-bound APC allows the localization of MACF1 to the cell membrane, which is required for microtubule capture and stabilization. Phosphorylates MACF1 and this phosphorylation inhibits the binding of MACF1 to microtubules which is critical for its role in bulge stem cell migration and skin wound repair.
Expressed in testis, thymus, prostate and ovary and weakly expressed in lung, brain and kidney.
Belongs to the protein kinase superfamily. CMGC Ser/Thr protein kinase family. GSK-3 subfamily. Contains 1 protein kinase domain.
Phosphorylated by AKT1 and ILK1. Activated by phosphorylation at Tyr-216.
Cytoplasm. Nucleus. Cell membrane. The phosphorylated form shows localization to cytoplasm and cell membrane. The MEMO1-RHOA-DIAPH1 signaling pathway controls localization of the phosophorylated form to the cell membrane.
Lane 1: Wild-type HAP1 cell lysate (20 µg) Lane 2: GSK3 beta knockout HAP1 cell lysate (20 µg) Lane 3: HeLa cell lysate (20 µg) Lane 4: A431 cell lysate (20 µg) Lanes 1 - 4: Merged signal (red and green). Green - ab73173 observed at 47 kDa. Red - loading control, ab8245, observed at 37 kDa.
ab73173 was shown to recognize GSK3 beta when GSK3 beta knockout samples were used, along with additional cross-reactive bands. Wild-type and GSK3 beta knockout samples were subjected to SDS-PAGE. ab73173 and ab8245 (loading control to GAPDH) were diluted at 1 µg/ml 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.
Western blot - Anti-GSK3 beta antibody (ab73173)
All lanes : Anti-GSK3 beta antibody (ab73173) at 1 µg/ml
Lane 1 : Jurkat Whole Cell Lysate - Staurosporine Treated (24hr, 500nM) Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
Performed under reducing conditions.
Predicted band size : 47 kDa Observed band size : 47 kDa
ICC/IF image of ab73173 stained MCF7 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab73173, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HeLa cells at 5µg/ml.
IHC image of GSK3 beta staining in human testis formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab71373, 5µg/ml, for 15 mins 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.
ICC/IF image of ab73173 stained HeLa cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab73173, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.
Western blot - GSK3 beta protein (Tagged) (ab60863)
Developed using the ECL technique
Performed under reducing conditions.
Predicted band size : 47 kDa
Exposure time : 90 secondsab60863 is a tagged recombinant protein with an expected molecular weight of 73kDa.