Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling Eg5 with ab254299 at 1/500 dilution, followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining in human tonsil (PMID:25277178) is observed.

The section was incubated with ab254299 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

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

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

All lanes : Anti-Eg5 antibody [EPR23277-60] (ab254299) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate
Lane 2 : MCF7 (human breast adenocarcinoma epithelial cell), whole cell lysate
Lane 3 : Jurkat (human T cell leukemia T lymphocyte), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution

Predicted band size: 119 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST. 

Exposure time:

Lanes 1-2: 15 seconds;
Lane 3: 5.5 seconds.

The expression profile observed is consistent with what has been described in the literature (PMID:23857769; 27492783).

Fresh lysates were tested in this WB.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (human cervix adenocarcinoma epithelial cell line) cells labelling Eg5 with ab254299 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HeLa cells. ab195889 Anti-alpha Tubulin antibody (Alexa Fluor^® 594) was used to counterstain tubulin at 1/100 dilution (Red). The Nuclear counterstain was DAPI (Blue).

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

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (Human cervix adenocarcinoma epithelial cell) cells labelling Eg5 with ab254299 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730) isotype control (Black) and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue).

Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

Eg5 was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell), whole cell lysate with ab254299 at 1/30 dilution. Western blot was performed on the immunoprecipitate using 254299 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa whole cell lysate 10 μg

Lane 2: ab254299 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254299 in HeLa whole cell lysate.

Blocking and dilution buffer and concentration: 5% NFDM/TBST.

Exposure time: 3 seconds

The minor bands beneath the target band are probably caused by degradation.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Eg5 with ab254299 at 1/500 dilution (1.18 μg/ml) followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining in human tonsil (PMID:25277178) is observed.

The section was incubated with ab254299 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

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

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).

All lanes : Anti-Eg5 antibody [EPR23277-60] (ab254299) at 1/1000 dilution

Lane 1 : Human testis tissue lysate
Lane 2 : Human tonsil tissue lysate
Lane 3 : Human lung tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : VeriBlot for IP Detection Reagent (HRP) (ab131366) at 1/1000 dilution

Predicted band size: 119 kDa
Observed band size: 130 kDa why is the actual band size different from the predicted?

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 15 seconds

The expression profile observed is consistent with what has been described in the literature (PMID:23857769; 27492783).

Negative control: Human lung (PMID: 27279560).

The minor bands beneath the target band are probably caused by degradation.

Immunohistochemical analysis of paraffin-embedded Human breast cancer tissue labeling Eg5 with ab254299 at 1/500 dilution followed by a ready to use Goat Anti-Rabbit IgG H&L (HRP) secondary antibody. Cytoplasmic staining in human tonsil (PMID:25277178) is observed. The section was incubated with ab254299 for 30 mins at room temperature.

The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

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

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is a ready to use Goat Anti-Rabbit IgG H&L (HRP).