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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 177 kDa (predicted molecular weight: 177 kDa).
Histone demethylase that specifically demethylates 'Lys-27' of histone H3, thereby playing a central role in histone code. Demethylates trimethylated and dimethylated H3 'Lys-27'. Plays a central role in regulation of posterior development, by regulating HOX gene expression. Involved in inflammatory response by participating in macrophage differentiation in case of inflammation by regulating gene expression and macrophage differentiation.
Belongs to the UTX family. Contains 1 JmjC domain.
ICC/IF image of ab169197 stained MCF-7 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 ab169197 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.
Western blot - Anti-KDM6B / JMJD3 antibody (ab169197)
All lanes : Anti-KDM6B / JMJD3 antibody (ab169197) at 1 µg/ml
Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate
Lysates/proteins at 10 µg per lane.
Secondary All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
Developed using the ECL technique.
Performed under reducing conditions.
Predicted band size: 177 kDa Observed band size: 177 kDa
Exposure time: 4 minutes
Due to the cellular localisation of KDM6B / JMJD3 an increase in expression is expected within the Nuclear fraction.
This blot was produced using a 3-8% Tris Acetate gel under the TA buffer system. The gel was run at 150V for 60 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with ab169197 overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution.