Anti-Gli3 antibody (ab6050)

Western blot - Gli3 antibody (ab6050)

Western blot for Gli3 antibody (ab6050) at 1/500 tested on the following tissue lysates:

Lane 1 : Human Brain
Lane 2:  Human Lung
Lane 3:  Human Spleen
Lane 4:  Mouse Brain
Lane 5:  Mouse Lung

Secondary ab: Goat anti-rabbit IgG ab6721 (1/5000)
Exposure time: 3 minutes
Expected molecular weight: 2 isoforms of Gli3 exist, one is the full length 170-190kDa and the other is a truncated isoform at ~80kDa. 
Cell lysates were loaded at 20µg per lane.

NB: This image shows the truncated isoform of Gli3 is detected by ab6050 in human lung (we do not know the identity of the 120-130kDa band).  It was not possible to detect Gli3 immunoreactivity by WB in the other lysates tested. This is likely due to low expression/abundance of Gli3 in these tissue lysates. Furthermore, Gli3 expression is likely to be developmentally regulated and induced, making it difficult to succesfully detect in whole tissue homogenates. The staining on the WB shows low background and minimal cross reactivity in whole homogenates. The 50kDa band is likely due to non-specific binding of secondary antibody.

Immunohistochemistry (Frozen sections) - Gli3 antibody (ab6050)

Gil3 expression in the radial glia of the developing mouse neocortex.

ab6050 Rabbit polyclonal to Gil3 on Frozen sections of mouse E12.5 whole brain, showing staing of the radial glia, of the developing neocortex. Sections were paraformaldehyde fixed, prior to heat mediated antigen retrieval in citric acid, and 14 hours incubation with ab6050 (1/100).

This image is courtesy of an Abreview submitted by Dr Dean Griffiths

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Gli3 antibody (ab6050)

ab6050 at 0.625 µg/ml staining human glioblastoma.

Immunocytochemistry/ Immunofluorescence-Gli3 antibody(ab6050)

ICC/IF image of ab6050 stained HepG2 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 (ab6050, 1µg/ml) overnight at +4ºC. 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.