Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

This WB data was generated using the same anti-Huntingtin antibody clone [EP867Y] in a different buffer formulation (cat# ab45169).

Lane 1: Wild type HAP1 whole cell lysate (20 µg)
Lane 2: Huntingtin knockout HAP1 whole cell lysate (20 µg)
Lane 3: SH-SY-5Y whole cell lysate (20 µg)
Lane 4: HeLa whole cell lysate (20 µg)

Lanes 1 - 4: Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab45169 was shown to recognize Huntingtin when Huntingtin knockout samples were used, along with additional cross-reactive bands. Wild-type and Huntingtin knockout samples were subjected to SDS-PAGE. ab45169 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C at 10000 dilution and 1/10000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

Overlay histogram showing SH-SY5Y cells stained with ab45169 (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 followed by the antibody (ab45169, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (ab96899) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) ( 1µg/1x10^6 cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in SH-SY5Y cells fixed with 4% paraformaldehyde/permeabilized in 0.1% PBS-Tween used under the same conditions.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45169).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45169).

ICC/IF image of ab45169 stained SKNSH 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 (ab45169, 1/200 dilution) 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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab45169).