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

This data was developed using the same antibody clone in a different buffer formulation (ab45169).

Lanes 1-4: Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - loading control ab7291 observed at 50 kDa.

ab45169 Anti-Huntingtin antibody [EP867Y] was shown to specifically react with Huntingtin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout sample ab256946 was used. Wild-type and Huntingtin knockout samples were subjected to SDS-PAGE. ab45169 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) were incubated overnight at 4°C at 1 in 10000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773)and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

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).

Ab45169 staining human Huntingtin in human brain tissue by immunohistochemistry using paraffin embedded tissue.

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

Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.

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).