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.

All lanes : Anti-SCA2 antibody [EPR23630-49] (ab254362) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : HEK-293T (human epithelial cell line from embryonic kidney transformed with large T antigen) whole cell lysate
Lane 3 : NIH/3T3 (mouse embryonic fibroblast) whole cell lysate
Lane 4 : PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 140 kDa
Observed band size: 145,41 kDa why is the actual band size different from the predicted?

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.

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.

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.