Recombinant 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.

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.

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.

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.

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.

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.

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.

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.

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

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.

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

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.

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.

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.