Anti-Huntingtin antibody [EP867Y]

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-Huntingtin antibody [EP867Y] (AB45169)

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.

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EP867Y] (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.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] (AB45169)

ab45169 staining human Huntingtin in human brain tissue by immunohistochemistry using paraffin embedded tissue.

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

• WB

Lab

Western blot - Anti-Huntingtin antibody [EP867Y] (AB45169)

ab45169 was shown to react with HTT in wild-type DMS53 cells in Western blot with loss of signal observed in a HTT knockout cell line. Wild-type DMS53 and HTT knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab45169 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/5000 dilution

Lane 1:

Wild-type DMS53 lysate at 30 µg

Lane 2:

HTT knock-out DMS53 lysate at 30 µg

false

• WB

Unknown

Western blot - Anti-Huntingtin antibody [EP867Y] (AB45169)

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 specifically recognize Huntingtin in wild-type HAP1 cells along with additional cross reactive bands. No band was observed when Huntingtin knockout samples were examined. 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 1/10,000 dilution and 1/10,000 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 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

Huntingtin knockout HAP1 whole cell lysate at 20 µg

Lane 3:

SH-SY5Y whole cell lysate at 20 µg

Lane 4:

HeLa whole cell lysate at 20 µg

Predicted band size: 348 kDa

false

• WB

Lab

Western blot - Anti-Huntingtin antibody [EP867Y] (AB45169)

ab45169 was shown to react with HTT in wild-type DMS53, HEK 293T and HAP1 cells in Western blot with loss of signal observed in a HTT knockout cell lines. Wild-type and HTT knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab45169 overnight at 4 °C at a 1/5000 dilution. Blots were incubated with secondary antibodies at 0.2 µg/mL before imaging.

These data were provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/5000 dilution

Lane 1:

Wild-type DMS53 lysate at 30 µg

Lane 2:

HTT knock-out DMS53 lysate at 30 µg

Lane 3:

Wild-type HEK 293T lysate at 30 µg

Lane 4:

HTT knock-out HEK 293T lysate at 30 µg

Lane 5:

Wild-type HAP1 lysate at 30 µg

Lane 6:

HTT knock-out HAP1 lysate at 30 µg

false

• WB

Lab

Western blot - Anti-Huntingtin antibody [EP867Y] (AB45169)

Lanes 1- 2 : Merged signal (red and green). Green - ab45169 observed at 348 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab45169 was shown to react with Huntingtin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line ab265976 (knockout cell lysate ab256946) was used. Wild-type HeLa and HTT knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab45169 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) overnight at 4°C at a 1 in 10000 dilution and a 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.

All lanes:

Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

Lane 1:

Wild-type HeLa cell lysate at 20 µg

Lane 2:

HTT knockout HeLa cell lysate at 20 µg

Predicted band size: 348 kDa

Observed band size: 348 kDa

false

• WB

Unknown

Western blot - Anti-Huntingtin antibody [EP867Y] (AB45169)

All lanes:

Western blot - Anti-Huntingtin antibody [EP867Y] (ab45169) at 1/10000 dilution

All lanes:

SH-SY-5Y cell lysate

Predicted band size: 348 kDa

Observed band size: 300 kDa

false