Anti-Huntingtin antibody [EPR5526]

• WB

Lab

Western blot - Anti-Huntingtin antibody [EPR5526] (AB109115)

Lanes 1- 2 : Merged signal (red and green). Green - ab109115 observed at 348 kDa. Red - Anti-alpha Tubulin antibody [DM1A] - Loading Control (ab7291) observed at 50 kDa.

ab109115 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. ab109115 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 [EPR5526] (ab109115) 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,43 kDa,52 kDa,55 kDa,56 kDa

Observed band size: 348 kDa

false

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized

SH-SY5Y (Human neuroblastoma from bone marrow cells) cells labeling Huntingtin with purified ab109115 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and cytoplasmic staining on SH-SY5Y cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows :
1. ab191472 at 1/1000 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemical analysis of paraffin-embedded Human astrocytoma labeling Huntingtin with purified ab109115 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500. Counter stained with Hematoxylin. Nuclear staining on cancer cells of astrocytoma.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemical analysis of paraffin-embedded Human cerebral cortex tissue labeling Huntingtin with purified ab109115 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500. Counter stained with Hematoxylin. Nuclear staining on neuron of human cerebral cortex was observed.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EPR5526] (AB109115)

ab109115 staining HTT in wild-type HeLa cells (top panel) and HTT knockout HeLa cells (bottom panel, available as ab265976). The cells were fixed with 4% formaldehyde (10 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 ab109115 at 1.0 µg/mL and ab7291 at 1.0 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) and goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) (shown in red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemical analysis of formalin fixed paraffin embedded human cerebrum staining Huntingtin with ab109115 at a concentration of 0.1 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab109115 anti-Huntingtin [EPR5526] was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EPR5526] (AB109115)

ab109115 staining HTT in wild-type HeLa cells (top panel) and HTT knockout HeLa cells (bottom panel, available as ab265976). 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 ab109115 at 1.0 µg/mL and ab7291 at 1.0 µg/mL overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (ab150081) (shown in green) and goat secondary antibody to Mouse IgG (Alexa Fluor® 594) (ab150120) (shown in red) both at 1/1000. Nuclear DNA was labelled with DAPI (shown in blue). Image was acquired with a high-content analyser (Operetta CLS, Perkin Elmer) and a maximum intensity projection of confocal sections is shown.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemical analysis of paraffin-embedded Mouse testis labeling Huntingtin with purified ab109115 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500. Counter stained with Hematoxylin. Cytoplasmic staining on spermatogenic cells of mouse testis.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• IHC-FoFr

Lab

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemistry (Frozen) analysis of mouse cerebrum tissue sections labeling Huntingtin with purified ab109115 at 1/100 (13.4 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/1000 (2 μg/ml) was used as the secondary antibody. Sections were fixed with 4% paraformaldehyde and permeabilised with 0.2% Triton X-100. Negative control : PBS instead of the primary antibody. DAPI (blue) was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized

Neuro-2a (Mouse neuroblastoma cells) cells labeling Huntingtin with purified ab109115 at 1/1000 dilution, followed by Goat anti-rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green).

Confocal image showing nuclear and cytoplasmic staining on Neuro-2a cell line.

The nuclear counter stain is DAPI (blue).

Tubulin is detected with ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution and ab150120 (AlexaFluor®594 Goat anti-Mouse secondary) at 1/1000 dilution (red).

The negative controls are as follows :
1. ab191472 at 1/1000 dilution followed by ab150120 (Alexa Fluor^® 594 Goat anti-Mouse secondary) at 1/1000 dilution.
2. ab7291 (anti-Tubulin mouse mAb) at 1/1000 dilution followed by ab150077 (Alexa Fluor^® 488 Goat Anti-Rabbit IgG H&L) at 1/1000 dilution.

• IHC-FoFr

Lab

Immunohistochemistry (PFA perfusion fixed frozen sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemistry (Frozen) analysis of mouse cerebellum tissue sections labeling Huntingtin with purified ab109115 at 1/100 (13.4 μg/ml). Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/1000 (2 μg/ml) was used as the secondary antibody. Sections were fixed with 4% paraformaldehyde and permeabilised with 0.2% Triton X-100. Negative control : PBS instead of the primary antibody. DAPI (blue) was used as nuclear counterstain. Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20) was performed.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Huntingtin antibody [EPR5526] (AB109115)

Immunohistochemical analysis of paraffin-embedded Rat testis labeling Huntingtin with purified ab109115 at 1/100 dilution followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500. Counter stained with Hematoxylin. Weak cytoplasmic staining on spermatogenic cells of rat testis.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• WB

Supplier Data

Western blot - Anti-Huntingtin antibody [EPR5526] (AB109115)

Blocking and Diluting buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-Huntingtin antibody [EPR5526] (ab109115) at 1/5000 dilution

Lane 1:

SH-SY5Y (Human neuroblastoma from bone marrow cells) whole cell lysate at 10 µg

Lane 2:

HeLa (Human epithelial cells from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 3:

PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysate at 10 µg

Lane 4:

Neuro-2a (Mouse neuroblastoma cells) whole cell lysate at 10 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/1000 dilution

Predicted band size: 348 kDa

Observed band size: 348 kDa

false

Exposure time: 1s

• WB

Lab

Western blot - Anti-Huntingtin antibody [EPR5526] (AB109115)

ab109115 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 ab109115 overnight at 4 °C at a 1/2000 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 [EPR5526] (ab109115) at 1/2000 dilution

Lane 1:

Wild-type DMS53 lysate at 30 µg

Lane 2:

HTT knock-out DMS53 lysate at 30 µg

false

• WB

Lab

Western blot - Anti-Huntingtin antibody [EPR5526] (AB109115)

Lanes 1 - 4 : Merged signal (red and green). Green - ab109115 observed at 348 kDa. Red - loading control, ab18058, observed at 130 kDa.

ab109115 was shown to specifically recognize HTT in wild-type HAP1 cells along with additional cross-reactive bands. No band was observed when HTT knockout samples were exmined. Wild-type and HTT knockout samples were subjected to SDS-PAGE. Unpurified ab109115 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-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Huntingtin antibody [EPR5526] (ab109115) at 1/10000 dilution

Lane 1:

Wild-type HAP1 whole cell lysate at 20 µg

Lane 2:

HTT 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

Supplier Data

Western blot - Anti-Huntingtin antibody [EPR5526] (AB109115)

Blocking and Diluting buffer and concentration : 5% NFDM /TBST

All lanes:

Western blot - Anti-Huntingtin antibody [EPR5526] (ab109115) at 1/50000 dilution

Lane 1:

Mouse brain lysate at 20 µg

Lane 2:

Rat brain lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/1000 dilution

Predicted band size: 348 kDa

Observed band size: 348 kDa

false

Exposure time: 30s