Recombinant Anti-SCA2 antibody [EPR23630-49] (ab254362)

ab254362 was shown to react with aTXN2 in wild-type HAP1 cells in Western blot with loss of signal observed in a ATXN2 knockout cell line. Wild-type HAP1 and ATXN2 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab254362 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with goat anti-rabbit HRP secondary antibodies at 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.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling SCA2 with ab254362 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on HeLa cell line. The nuclear counterstain is DAPI (blue).
Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

The negative control is secondary antibody only.

The molecular weight observed is consistent with what has been described in the literature (PMID: 9989626).
Lysates should be made freshly and used in WB immediately to minimize protein degradation.

Blocking/Dilution buffer: 5% NFDM/TBST.

Exposure times: Lanes 1-3: 10 seconds; Lane 4: 15 seconds.

Immunohistochemical analysis of paraffin-embedded human cerebrum tissue labeling SCA2 with ab254362 at 1/1000 dilution, followed by a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on neurons of human cerebrum (PMID: 9989626). The section was incubated with ab254362 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized HeLa (human epithelial cell line from cervix adenocarcinoma) cells labeling SCA2 with ab254362 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). 

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody. 

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebellum tissue labeling SCA2 with ab254362 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. Positive staining on mouse cerebellum is observed. Nuclear counterstain is DAPI.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. 

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded rat cerebellum tissue labeling SCA2 with ab254362 at 1/1000 dilution, followed by a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in rat cerebellum (PMID: 26868665). The section was incubated with ab254362 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Intracellular flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling SCA2 with ab254362 at 1/500 dilution (red) compared with a Rabbit IgG, monoclonal [EPR25A] - Isotype Control (ab172730) (black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (blue). 

Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/2000 dilution was used as the secondary antibody. 

Immunohistochemical analysis of paraffin-embedded mouse cerebellum tissue labeling SCA2 with ab254362 at 1/1000 dilution, followed by a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining in mouse cerebellum (PMID: 26868665). The section was incubated with ab254362 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunohistochemical analysis of 4% PFA-fixed, 0.2% Triton X-100 permeabilized frozen mouse cerebrum tissue labeling SCA2 with ab254362 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. Positive staining on mouse cerebrum is observed. Nuclear counterstain is DAPI.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at a 1/1000 dilution. 

Heat mediated antigen retrieval using sodium citrate buffer (10mM citrate pH 6.0 + 0.05% Tween-20).

Immunohistochemical analysis of paraffin-embedded human cerebellum tissue labeling SCA2 with ab254362 at 1/1000 dilution, followed by a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Cytoplasmic staining on Purkinje cells in human cerebellum (PMID: 9989626). The section was incubated with ab254362 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use secondary from Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (mouse embryo fibroblast cell line) cells labeling SCA2 with ab254362 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplasmic staining on NIH/3T3 cell line. The nuclear counterstain is DAPI (blue).
Tubulin is detected with ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) at 1/200 dilution (red).

The negative control is secondary antibody only.