Anti-NSMase2 antibody

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NSMase2 antibody (AB85017)

ICC/IF image of ab85017 stained B35 cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with the antibody (ab85017, 5μg/ml) overnight at +4°C and ab195887, Mouse monoclonal to alpha Tubulin (Alexa Fluor^® 488), at a 1/250 dilution (shown in green). The secondary antibody (shown in red) was Alexa Fluor® 647 goat anti-rabbit IgG (H+L) ab150083 used at a 1/1000 dilution for 1h. Nuclear DNA was labelled with DAPI (shown in blue).

Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-NSMase2 antibody (AB85017)

ICC/IF image of ab85017 stained PC12 cells. The cells were 4% PFA 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 (ab85017, 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) PC12 cells at 5µg/ml.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-NSMase2 antibody (AB85017)

ab85017 staining Sphingomyelin phosphodiesterase 3 in primary hippocampal rat neurons/glia, (obtained from Neuromics, cat. no. PC35101), DIV14. cells. The cells were fixed with 100% methanol (5 min), permeabilized with 0.1% PBS-Tween for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated overnight at 4°C with ab85017 at 5µg/ml and ab7291, Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control. Cells were then incubated with ab150081, Goat polyclonal Secondary Antibody to Rabbit IgG - H&L (Alexa Fluor^® 488), pre-adsorbed at 1/1000 dilution (shown in green) and ab150120, Goat polyclonal Secondary Antibody to Mouse IgG - H&L (Alexa Fluor^® 594), pre-adsorbed at 1/1000 dilution (shown in pseudocolour red). Nuclear DNA was labelled with DAPI (shown in blue).

Image was acquired with a confocal microscope (Leica-Microsystems TCS SP8) and a single confocal section is shown.

• WB

Unknown

Western blot - Anti-NSMase2 antibody (AB85017)

All lanes:

Western blot - Anti-NSMase2 antibody (ab85017) at 1 µg/mL

All lanes:

Brain (Rat) Tissue Lysate at 10 µg

Secondary

All lanes:

Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Predicted band size: 71 kDa

Observed band size: 102 kDa,71 kDa

true

Exposure time: 20min