All lanes : Anti-MiTF antibody [D5] (ab3201) at 5 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) whole cell lysate
Lane 2 : HeLa nuclear lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 59 kDa
Observed band size: 40 kDa why is the actual band size different from the predicted?


Exposure time: 10 minutes

ab3201 staining MiTF in the SK-MEL-28 (Human cutaneous melanoma cell line) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with Paraformaldehyde and permeabilized with Triton X-100 0.5% in PBS. Samples were incubated with primary antibody (1/100) for 24 hours at 4°C. An Alexa Fluor^®488-conjugated Goat anti-mouse IgG polyclonal(1/1000) was used as the secondary antibody. SK-MEL-28 cells was grown on chamber slide. MiTF was found to strictly localised in the nucleus. Cells were counter-stained with DAPI.

See Abreview

ICC/IF image of ab3201 stained Malme-3M (Human melanoma cell line) 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 ab3201 at 5µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- mouse (ab150117) 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.

Overlay histogram showing Malme-3 (Human skin fibroblast cell line) cells stained with ab3201 (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. The cells were then incubated with the antibody (ab3201, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2µg/1x10⁶ cells ) used under the same conditions. Acquisition of >5,000 events was performed.