Anti-Huntingtin antibody [EP867Y] - BSA and Azide free

7 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EP867Y] - BSA and Azide free (ab225573)

  Anti-Huntingtin antibody [EP867Y] 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 (Anti-Huntingtin antibody [EP867Y] ab45169).

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

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

  This data was developed using the same antibody clone in a different buffer formulation (Anti-Huntingtin antibody [EP867Y] ab45169).
  

  Lanes 1- 2: Merged signal (red and green). Green - Anti-Huntingtin antibody [EP867Y] ab45169 observed at 348 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291) observed at 50 kDa.

  Anti-Huntingtin antibody [EP867Y] ab45169 was shown to react with Huntingtin in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human HTT (Huntingtin) knockout HeLa cell line ab265976 (knockout cell lysate Human HTT (Huntingtin) knockout HeLa 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. Anti-Huntingtin antibody [EP867Y] ab45169 and Anti-alpha Tubulin antibody [DM1A] - Loading Control (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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (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] (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

  Performed under reducing conditions.

  Predicted band size: 348 kDa

  Observed band size: 348 kDa

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

  ICC/IF image of Anti-Huntingtin antibody [EP867Y] 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 (Anti-Huntingtin antibody [EP867Y] ab45169, 1/200 dilution) overnight at +4°C. The secondary antibody (green) was Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed 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 (Anti-Huntingtin antibody [EP867Y] ab45169).

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  Western blot - 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# Anti-Huntingtin antibody [EP867Y] ab45169).
  

  Lanes 1 - 4: Merged signal (red and green). Green - Anti-Huntingtin antibody [EP867Y] ab45169 observed at 348 kDa. Red - loading control, ab18058, observed at 130 kDa.

  Anti-Huntingtin antibody [EP867Y] 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. Anti-Huntingtin antibody [EP867Y] ab45169 and ab18058 (Mouse anti-Vinculin loading control) were incubated overnight at 4°C both at 1/10000 dilution. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777 secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Huntingtin antibody [EP867Y] (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

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

  Overlay histogram showing SH-SY5Y cells stained with Anti-Huntingtin antibody [EP867Y] 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 (Anti-Huntingtin antibody [EP867Y] ab45169, 1/100 dilution) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-rabbit IgG (H+L) (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed 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 (Anti-Huntingtin antibody [EP867Y] ab45169).

• 

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

  This data was developed using Anti-Huntingtin antibody [EP867Y] ab45169, the same antibody clone in a different buffer formulation.

  Anti-Huntingtin antibody [EP867Y] 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 Anti-Huntingtin antibody [EP867Y] 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] (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

• 

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

  This data was developed using Anti-Huntingtin antibody [EP867Y] ab45169, the same antibody clone in a different buffer formulation.

  Anti-Huntingtin antibody [EP867Y] 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 Anti-Huntingtin antibody [EP867Y] 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] (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